Mammalian cell culture-produced neublastin antibodies

ABSTRACT

The present disclosure pertains to a mammalian cell culture genetically modified to express, and which expresses, a neublastin antibody polypeptide, or fragment thereof, in the culture, and to a neublastin antibody polypeptide, or fragment thereof, made by a mammalian cell culture genetically modified to express, and which expresses, the neublastin antibody polypeptide, or fragment thereof.

This application is a continuation-in-part of U.S. application Ser. No.15/291,257 filed Oct. 12, 2016, which is a continuation of U.S.application Ser. No. 14/760,182 filed Jul. 9, 2015, which is a 35 U.S.C.371 national stage application of PCT/US14/11130 filed Jan. 10, 2014,which claims the benefit of U.S. provisional application No. 61/751,067filed Jan. 10, 2013, the disclosures of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention pertains to a cell culture medium comprisingdextran sulfate or a mixture of dextran sulfate and ferric citrate, andmethods of use. The present invention further pertains to a method ofproducing a protein of interest in a large-scale cell culture, a methodof producing neublastin antibodies in mammalian cells, and to neublastinantibodies produced in mammalian cell culture.

BACKGROUND

Over the last few decades, much research has focused on the productionof recombinant proteins, e.g. monoclonal antibodies, and the work hastaken a variety of angles. While much work in the literature hasutilized media containing sera or hydrolysates, chemically defined mediawere also developed in order to eliminate the problematic lot-to-lotvariation of complex components (Luo and Chen, Biotechnol. Bioeng.97(6):1654-165.9 (2007)). An improved understanding of the cell culturehas permitted a shift to chemically defined medium without compromisingon growth, viability, titer, etc., To date optimized chemically definedprocesses have been reported with titers as high as 7.5-10 g/L (Huang etal., Biotechnology Progress 26(5):1400-1410 (2010); Ma et at,Biotechnol. Prog. 25(5):1353-1363 (2009); Yu et al.; Biotechnol. Bioeng.108(5):1078-1088 (2011)). In general, the high titer chemically definedprocesses are fed batch processes with cultivation times of 11-18 days.The process intensification has been achieved without compromisingproduct quality while maintaining relatively high viabilities.

Achievement of a robust, scalable production process includes more thanincreasing the product titer while maintaining high product quality. Theprocess must also predictably require the main carbohydrate source suchthat the feeding strategy does not need to change across scales. As manyprocesses use glucose as the main carbohydrate, and have lactate andammonium as the main byproducts, the time course of these three criticalchemicals should also scale.

A recent metabolomics study performed by Ma and coworkers (Ma et al.,Biotechnol. Prog. 25(5):1353-1363 (2009)) suggested a blockage in theTCA cycle, resulting in an early phase secretion of citrate and latercitrate consumption. The process used by Ma may also have subsequentlyresulted in high LPR: if the viability permitted further extension ofthe process. The feeding of pyruvate (0.02 M) was shown to increaseantibody production by 43% in a continuous culture of a hybridoma cell(Omasa et al., Bioproc. Biosys. Engin. 33(1):117-125 (2010)). Thefeeding of citrate (0.05 M and 0.01 M) in the same culture systemresulted only in a ˜5-10% increase in antibody production. Bai recentlyreported increased antibody production in a chemically defined CHO cellculture supplemented with a combination of high concentrations ofchemically defined iron and high concentrations of citrate (Bai et al.,Biotechnol. Prog. 27(1):209-219 (2011)). Citrate supplementation alone,however, could not support stable cell growth at all.

There is a need in the art to further improve recombinant proteinproduction processes to eliminate lot-to-lot metabolic variability.Provided herein are compositions and methods to prevent or reducemetabolic variability encountered in recombinant protein producing cellcultures.

SUMMARY OF THE INVENTION

The present invention pertains to a method of culturing cells in amedium comprising supplementing the medium with a feed comprising asufficient amount of dextran sulfate. The present invention alsopertains to a method of culturing cells in a medium comprising orsupplementing the medium with a feed comprising a mixture of dextransulfate and ferric citrate.

In one embodiment, the medium and/or feed comprise dextran sulfate in anamount sufficient to increase the dextran sulfate concentration in themedium by between about 0.1 g/L and about 5 g/L. In another embodiment,the medium and/or feed comprise ferric citrate in an amount sufficientto raise the ferric citrate concentration in the medium about 1 mM andabout 50 mM.

The present invention also pertains to a cell culture medium comprisingdextran sulfate or a mixture of dextran sulfate and ferric citrate.

The present invention further provides a cell culture compositioncomprising cells capable of expressing a polypeptide of interest and amedium comprising dextran sulfate or a mixture of dextran sulfate andferric citrate.

The present invention further pertains to a conditioned cell culturemedium produced by a method disclosed herein. In one embodiment, theconditioned medium comprises a polypeptide of interest produced by amethod disclosed herein. In a specific embodiment, a conditioned mediumaccording to the present invention comprises an antibody. In anotherspecific embodiment, a conditioned medium according to the presentinvention comprises a Transforming Growth Factor (TGF) beta superfamilysignaling molecule. In yet another specific embodiment, a conditionedmedium according to the present invention comprises a blood clottingfactor.

In still another aspect, the disclosure provides a cell culturecomprising a mammalian cell line that has been genetically modified toexpress a neublastin antibody polypeptide, or a fragment thereof, in thecell culture medium. In certain embodiments, the mammalian cell line isa Chinese Hamster Ovary (CHO) cell line. In some embodiments, theneublastin antibody polypeptide, or fragment thereof, comprises SEQ IDNO:2, or a portion thereof, and/or SEQ ID NO:4, or a portiont thereof.In particular embodiments, the cells have been adapted to grow in aserum-free medium, an animal protein-free medium, or a chemicallydefined medium.

In some embodiments, the mammalian cell culture is a perfusion cultureor is a fed batch culture. In certain embodiments, the cell culturemedium is a serum-free medium, an animal protein-free medium, or achemically defined medium.

In yet another aspect, the disclosure provides a neublastin antibodypolypeptide, or fragment thereof, produced in a mammalian cell culture,the culture comprising mammalian cells genetically modified to express,and which express, the neublastin antibody polypeptide, or a fragmentthereto, in the mammalian cell culture. In some embodiments, themammalian cell line is a CHO cell line. In some embodiments, theneublastin antibody polypeptide, or fragment thereof, comprises SEQ IDNO:2, or a portion thereof, and/or SEQ ID NO:4, or a portion thereof. Inparticular embodiments, the cells have been adapted to grow inserum-free medium, an animal protein-free medium, or a chemicallydefined medium. In some embodiments, the neublastin antibodypolypeptide, or fragment thereof, has been isolated from the mammaliancell culture.

In another aspect, the disclosure provides a pharmaceutical formulationcomprising a neublastin antibody polypeptide, or fragment thereof, whichhas been isolated from a mammalian cell culture comprising mammaliancells genetically modified to express the neublastin antibodypolypeptide, or a fragment thereto, in the mammalian cell culture. Insome embodiments, the mammalian cell culture is a CHO cell culture. Inparticular embodiments, the neublastin antibody polypeptide comprisesSEQ ID NO:2, or portion thereof, and/or SEQ ID NO:4, or a portionthereof.

BRIEF DESCRIPTION OF THE DRAWING

The foregoing and other objects of the present disclosure, the variousfeatures thereof, as well as the disclosure itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings in which:

FIGS. 1A-1B are graphic representations of the citrate (A) and ammoniumproduction (B) after addition of ferric citrate and dextran sulfatemaintains lactate levels and decreases ammonium production;

FIG. 2A-2B are graphic representations of the VC/ML (A) and viability(B) after addition of dextran sulfate to shaker flasks;

FIG. 3A-3B are graphic representations of the VCD (A) and viability ofcultures after the addition of dextran sulfate to the bioreactorinoculum train culture;

FIG. 4A-4B are graphic representations of viable cell density (A) andthe viability (B) of cell cultures in production bioreactors inoculatedusing dextran sulfate;

FIG. 5A is a schematic representation of the amino acid sequence of theheavy chain of the neublastin antibody including a signal peptide;

FIG. 5B is a schematic representation of the amino acid sequence of theheavy chain of the neublastin antibody;

FIG. 5C is a schematic representation of the amino acid sequence of thelight chain of the neublastin antibody including a signal peptide;

FIG. 5D is a schematic representation of the amino acid sequence of thelight chain of the neublastin antibody;

FIG. 6 is a representation of a representation elusion profile ofneublastin antibody on a size exclusion chromatography (SEC) column;

FIG. 7 is a representation of a polyacrylamide gel of the peak materialfrom the SEC column shown in FIG. 6;

FIG. 8A is a schematic representation (SEQ ID NO: 5) of the neublastinheavy chain with signal peptide starting “MDSRLNLVFL” (residues 1 to 10of SEQ ID NO: 1);

FIG. 8B is a schematic representation (SEQ ID NO: 6) of the degeneratereverse translation of the neublastin heavy chain;

FIG. 8C is a schematic representation (SEQ ID NO: 7) of the reversetranslation of the neublastin heavy chain with no signal peptidestarting “EVKVVESGGG” (residues 1 to 10 of SEQ ID NO: 2) showing themost likely codons;

FIG. 8D is a schematic representation (SEQ ID NO: 8) of the degeneratereverse translation of the neublastin heavy chain;

FIG. 8E is a schematic representation (SEQ ID NO: 9) of the reversetranslation of the neublastin light chain with the signal peptidestarting “MKSQTQVFVF” (residues 1 to 10 of SEQ ID NO: 3);

FIG. 8F is a schematic representation (SEQ ID NO: 10) of the degeneratereverse translation of the neublastin light chain;

FIG. 8G is a schematic representation (SEQ ID NO: 11) of the reversetranslation of the neublastin light chain starting “SIVMTQTPKF”(residues 1 to 10 of SEQ ID NO: 4) showing the most likely codons; and

FIG. 8H is a schematic representation (SEQ ID NO: 12) of the degeneratereverse translation of the neublastin light chain.

DETAILED DESCRIPTION

All documents, articles, publications, patents, and patent applicationsmentioned in this specification are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. The initial definitionprovided for a group or term herein applies to that group or termthroughout the present specification individually or as part of anothergroup, unless otherwise indicated.

I. Definitions

The term “antibody” is used to mean an immunoglobulin molecule thatrecognizes and specifically binds to a target or antigen, such as aprotein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing etc., through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. As used, herein, the term encompasses intact polyclonalantibodies, intact monoclonal antibodies, antibody fragments (such asFab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) mutants,multispecific antibodies such as bispecific antibodies generated from atleast two intact antibodies, monovalent or monospecific antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively.

As used herein, the term “antibody fragment” refers to a portion of anintact antibody and refers to the antigenic determining variable regionsof an intact antibody. Examples of antibody fragments include, but arenot limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies,single chain antibodies, and multispecific antibodies formed fromantibody fragments.

The term “basal media formulation” or “basal media” as used hereinrefers to any cell culture media used to culture cells that has not beenmodified either by supplementation, or by selective removal of a certaincomponent.

The term “batch culture” as used herein refers to a method of culturingcells in which all the components that will ultimately be used inculturing the cells, including the medium (see definition of “medium”below) as well as the cells themselves, are provided at the beginning ofthe culturing process. A batch culture is typically stopped at somepoint and the cells and/or components in the medium are harvested andoptionally purified.

The term “bioreactor” as used herein refers to any vessel used for thegrowth of a mammalian cell culture. The bioreactor can be of any size solong as it is useful for the culturing of mammalian cells. Typically,the bioreactor will be at least 1 titer and can be 10, 50, 100, 250,500, 1000, 2000, 2,500, 3000, 5000, 8000, 10,000, 12,000, 15,000,20,000, 30,000 liters or more, or any volume in between. For example, abioreactor will be 10 to 5,000 liters, 10 to 10,000 liters, 10 to 15,000liters, 10 to 20,000 liters, 10 to 30,000 liters, 50 to 5,000 liters, 50to 10,000 liters, 50 to 15,000 liters, 50 to 20,000 liters, 50 to 30,000liters, 1,000 to 5,000 liters, or 1,000 to 3,000 liters. The internalconditions of the bioreactor, including, but not limited to pH andtemperature, are typically controlled during the culturing period. Thebioreactor can be composed of any material that is suitable for holdingmammalian cell cultures suspended in media under the culture conditionsof the present invention, including glass, plastic or metal. The term“production bioreactor” as used herein refers to the final bioreactorused in the production of the polypeptide or protein of interest. Thevolume of the large-scale cell culture production bioreactor istypically at least 500 liters and can be 1000, 2000, 2500, 5000, 8000,10,000, 12,0000, 15,000 liters or more, or any volume in between. Forexample, the large scale cell culture reactor will be between about 500liters and about 20,000 liters, about 500 liters and about 10,000liters, about 500 liters and about 5,000 liters, about 1,000 liters andabout 30,000 liters, about 2,000 liters and about 30,000 liters, about3,000 liters and about 30,000 liters, about 5,000 liters and about30,000 liters, or about 10,000 liters and about 30,000 liters, or alarge scale cell culture reactor will be at least about 500 liters, atleast about 1,000 liters, at least about 2,000 liters, at least about3,000 liters, at least about 5,000 liters, at least about 10,000 liters;at least about 15,000 liters, or at least about 20,000 liters. One ofordinary skill in the art will be aware of and will be able to choosesuitable bioreactors for use in practicing, the present invention.

The term “cell density” as used herein refers to that number of cellspresent in a given volume of medium.

The terms “culture”, “cell culture”, and “eukaryotic cell culture” asused herein refer to a eukaryotic cell population that is suspended in amedium (see definition of “medium” below) under conditions suitable tosurvival and/or growth of the cell population. As will be clear to thoseof ordinary skill in the art, these terms as used herein can refer tothe combination comprising the mammalian cell population and the mediumin which the population is suspended.

The term “fed-batch culture” as used herein refers to a method ofculturing cells in which additional components are provided to theculture at some time subsequent to the beginning of the culture process.A fed-batch culture can be started using a basal medium. The culturemedium with which additional components are provided to the culture atsome time subsequent to the beginning of the culture process is a feedmedium. The provided components typically comprise nutritionalsupplements for the cells which have been depleted during the culturingprocess. In one embodiment, a feed medium described herein comprisesdextran sulfate or a mixture of dextran sulfate and ferric citrate. Inanother embodiment, a feed medium described herein consists of dextransulfate or a mixture of dextran sulfate and ferric citrate. A fed-batchculture is typically stopped at some point and the cells and/orcomponents in the medium are harvested and optionally purified.

“Growth phase” of the cell culture refers to the period of exponentialcell growth (the log phase) where cells are generally rapidly dividing.During this phase, cells are cultured for a period, usually between 1-4days, and under such conditions that cell growth is maximized. Thedetermination of the growth cycle for the host cell can be determinedfor the host cell envisioned without undue experimentation. “Period oftime and under such conditions that cell growth is maximized” and thelike, refer to those culture conditions that, for a cell line, aredetermined to be optimal for cell growth and division. During the growthphase, cells are cultured in nutrient medium containing the necessaryadditives generally at about 25°-40° C., in a humidified, controlledatmosphere, such that optimal growth is achieved for the cell line.Cells are maintained in the growth phase for a period of about betweenone and four days, usually between two to three days. The length of thegrowth phase for the cells can be determined without undueexperimentation. For example, the length of the growth phase will be theperiod of time sufficient to allow the particular cells to reproduce toa viable cell density within a range of about 20%-80% of the maximalpossible viable cell density if the culture was maintained under thegrowth conditions.

“Production phase” of the cell culture refers to the period of timeduring which cell growth has plateaued. During the production phase,logarithmic cell growth has ended and protein production is primary.During this period of time the medium is generally supplemented tosupport continued protein production and to achieve the desiredglycoprotein product.

The term “expression” or “expresses” are used herein to refer totranscription and translation occurring within a host cell. The level ofexpression of a product gene in a host cell can be determined on thebasis of either the amount of corresponding mRNA that is present in thecell or the amount of the protein encoded by the product gene that isproduced by the cell. For example, mRNA transcribed from a product geneis desirably quantitated by northern hybridization. Sambrook et al.,Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring HarborLaboratory Press, 1989). Protein encoded by a product gene can bequantitated either by assaying for the biological activity of theprotein or by employing assays that are independent of such activity,such as western blotting or radioimmunoassay using antibodies that arecapable of reacting with the protein. Sambrook et al., MolecularCloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring HarborLaboratory Press, 1989).

The term “hybridoma” as used herein refers to a cell created by fusionof an immortalized cell derived from an immunologic source and anantibody-producing cell. The resulting hybridoma is an immortalized cellthat produces antibodies. The individual cells used to create thehybridoma can be from any mammalian source, including, but not limitedto, rat, pig, rabbit, sheep, pig, goat, and human. The term alsoencompasses trioma cell lines, which result when progeny of heterohybridmyeloma fusions, which are the product of a fusion between human cellsand a murine myeloma cell line, are subsequently fused with a plasmacell. Furthermore, the term is meant to include any immortalized hybridcell line that produces antibodies such as, for example, quadromas (See,e.g., Milstein et al., Nature, 537:3053 (1983).

The terms “medium”, “cell culture medium”, “culture medium”, and “growthmedium” as used herein refer to a solution containing nutrients whichnourish growing eukaryotic cells. Typically, these solutions provideessential and non-essential amino acids, vitamins, energy sources,lipids, and trace elements required by the cell for minimal growthand/or survival. The solution can also contain components that enhancegrowth and/or survival above the minimal rate, including hormones andgrowth factors. The solution is formulated to a pH and saltconcentration optimal for cell survival and proliferation. The mediumcan also be a “defined medium” or “chemically defined medium” aserum-free medium that contains no proteins, hydrolysates or componentsof unknown composition. Defined media are free of animal-derivedcomponents and all components have a known chemical structure. One ofskill in the art understands a defined medium can comprise recombinantpolypeptides or proteins, for example, but not limited to, hormones,cytokines, interleukins and other signaling molecules.

The term “perfusion culture” as used herein refers to a method ofculturing cells in which additional components are provided continuouslyor semi-continuously to the culture subsequent to the beginning of theculture process. The provided components-typically comprise nutritionalsupplements for the cells which have been depleted during the culturingprocess. A portion of the cells and/or components in the medium aretypically harvested on a continuous or semi-continuous basis and areoptionally purified.

The terms “polypeptide” or “protein” as used herein refers a sequentialchain of amino acids linked together via peptide bonds. The term is usedto refer to an amino acid chain of any length, but one of ordinary skillin the art will understand that the term is not limited to lengthychains and can refer to a minimal chain comprising two amino acidslinked together via a peptide bond. If a single polypeptide is thediscrete functioning unit and does require permanent physicalassociation with other polypeptides in order to form the discretefunctioning unit, the terms “polypeptide” and “protein” as used hereinare used interchangeably. If discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” as used herein refers to the multiple polypeptidesthat are physically coupled and function together as the discrete unit.

“Recombinantly expressed polypeptide” and “recombinant polypeptide” asused herein refer to a polypeptide expressed from a host cell that hasbeen genetically engineered to express that polypeptide. Therecombinantly expressed polypeptide can be identical or similar topolypeptides that are normally expressed in the mammalian host cell. Therecombinantly expressed polypeptide can also foreign to the host cell,i.e. heterologous to peptides normally expressed in the mammalian hostcell. Alternatively, the recombinantly expressed polypeptide can bechimeric in that portions of the polypeptide that contain amino acidsequences that are identical or similar to polypeptides normallyexpressed in the mammalian host cell, while other portions are foreignto the host cell. As used herein, the terms “recombinantly expressedpolypeptide” and “recombinant polypeptide” also encompasses an antibodyproduced by a hybridoma.

The term “seeding” as used herein refers to the process of providing acell culture to a bioreactor or another vessel. In one embodiment, thecells have been propagated previously in another bioreactor or vessel.In another embodiment, the cells have been frozen and thawed immediatelyprior to providing them to the bioreactor or vessel. The term refers toany number of cells, including a single cell.

The term “titer” as used herein refers to the total amount ofrecombinantly expressed polypeptide or protein produced by a cellculture divided by a given amount of medium volume. Titer is typicallyexpressed in units of milligrams of polypeptide or protein permilliliter of medium or in units of grams of polypeptide or protein perliter of medium.

As used in the present disclosure and claims, the singular forms “a”,“an”, and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that whenever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting” and/or “consisting essentially of” are also provided.

II. Cell Culture Medium and Methods of Using the Same

The present invention relates to cell culture media and methods of usethereof. The media of the invention reduces lot-to-lot metabolicvariability associated with a metabolic shift to lactate production. Amedium according to the invention can be used in a batch culture,fed-batch culture or a perfusion culture. In one embodiment, a medium ofthe invention is a basal medium. In another embodiment, a medium of theinvention is a feed medium.

In one embodiment, a medium according to the present invention comprisesdextran sulfate. A medium can comprise sufficient amount of dextransulfate to increase the dextran sulfate concentration in the culture bybetween about 0.01 g/L and about 5 g/L. In one embodiment, a feed mediumdescribed herein comprises sufficient amount of dextran sulfate toincrease the dextran sulfate concentration in the culture by betweenabout 0.01 g/L and about 5 g/L, about 0.01 g/L and about 4 g/L, about0.01 g/L and about 3 g/L, about 0.01 g/L and about 2 g/L, about 0.01 g/Land about 1 g/L, about 0.01 g/L and about 0.5 g/L, about 0.01 g/L andabout 0.25 g/L, about 0.05 g/L and about 5 g/L, about 0.05 g/L and about4 g/L, about 0.05 g/L and about 3 g/L, about 0.05 g/L and about 2 g/L,about 0.05 g/L and about 1 g/L, about 0.05 g/L and about 0.5 g/L, about0.05 g/L and about 0.25 g/L, about 0.1 g/L and about 5 g/L, about 0.1g/L and about 4 g/L, about 0.1 g/L and about 3 g/L, about 0.1 g/L andabout 2 g/L, about 0.1 g/L and about 1 g/L, about 0.1 g/L and about 0.5g/L, about 0.1 g/L and about 0.25 g/L, about 0.2 g/L and about 5 g/L,about 0.2 g/L and about 4 g/L, about 0.2 g/L, and about 3 g/L, about 0.2g/L, and about 2 g/L, about 0.2 g/L and about 1 g/L, about 0.2 g/L andabout 0.5 g/L, about 0.2 g/L and about 0.25 g/L, about 0.25 g/L andabout 5 g/L, about 0.25 g/L and about 4 g/L, about 0.25 g/L and about 3g/L, about 0.25 g/L and about 2 g/L, about 0.25 g/L and about 1 g/L, orabout 0.25 g/L and about 0.5 g/L. In another embodiment, a feed mediumdescribed herein comprises sufficient amount of dextran sulfate toincrease the dextran sulfate concentration in the culture by about 0.01g/L, about 0.02 g/L, about 0.03 g/L, about 0.04 g/L, about 0.05 g/L,about 0.06 g/L, about 0.07 g/L, about 0.08 g/L, about 0.09 g/L, about0.1 g/L, about 0.15 g/L, about 0.2 g/L, about 0.25 g/L, about 0.5 g/L,about 0.6 g/L, about 0.7 g/L, about 0.8 g/L, about 0.9 g/L, about 1 g/L,about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g/L,about 4 g/L, about 4.5 g/L, or about 5 g/L. A skilled artisan readilyunderstands that the absolute amount of dextran sulfate supplemented bya feed medium to a cell culture can be calculated from the volume offeed medium added to the culture and the dextran sulfate concentrationof the feed medium.

In one embodiment, a medium according to the present invention comprisesa mixture of dextran sulfate and ferric citrate. A medium can comprisesufficient amount of ferric citrate to increase the ferric citrateconcentration in the culture by between about 1 mM and about 50 mM. Inone embodiment, a feed medium described herein comprises sufficientamount of ferric citrate to increase the ferric citrate concentration inthe culture by between about 1 mM and about 50 mM, about 1 mM and about40 mM, about 1 mM and about 35 mM, about 1 mM and about 30 mM, about 1mM and about 25 mM, about 1 mM and about 20 mM, about 1 mM and about 15mM, about 1 mM and about 14 mM, about 1 mM and about 13 mM, about 1 mMand about 12 mM, about 1 mM and about 11 mM, about 1 mM and about 10 mM,about 2 mM and about 50 mM, about 3 mM and about 50 mM, about 4 mM andabout 50 mM, about 5 mM and about 50 mM, about 10 mM and about 50 mM,about 15 mM and about 50 mM, about 20 mM and about 50 mM, or about 30 mMand about 50 mM. In another embodiment, a feed medium described hereincomprises sufficient amount of ferric citrate to increase the ferriccitrate concentration in the culture by about 1 mM, about 2 mM, about 3mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM,about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM,about 30 mM, about 35 mM, about 40, about 45 mM or about 50 mM. Askilled artisan readily understands that the absolute amount of ferriccitrate supplemented by a feed medium to a cell culture can becalculated from the volume of feed medium added to the culture and theferric citrate concentration of the feed medium.

In one embodiment, a medium described herein is a serum-free medium,animal protein-free medium or a chemically-defined medium. In a specificembodiment, a medium described herein is a chemically-defined medium.

The present invention further provides a cell culture compositioncomprising a medium described herein and cells.

In one embodiment, a cell culture composition according to the inventioncan be a batch culture, fed-batch culture or a perfusion culture. In aspecific embodiment, a cell culture composition of the invention is afed batch culture.

In one embodiment, a cell culture composition described herein comprisesmammalian cells such as, but not limited to, CHO cells, HEK cells, NSOcells, PER.C6 cells, 293 cells, HeLa cells, and MDCK cells. In aspecific embodiment, a cell culture composition described hereincomprises CHO cells. In another specific embodiment, a cell culturecomposition described herein comprises HEK cells. In another specificembodiment, a-cell culture composition described herein compriseshybridoma cells.

A cell culture composition described herein can comprise cells that havebeen adapted to grow in serum free medium, animal protein free medium orchemically defined medium. Alternatively, it can comprise cells thathave been genetically modified to increase their life-span in culture.In one embodiment, the cells have been modified to express ananti-apoptotic gene. In a specific embodiment, the cells have beenmodified to express the bcl-xL antiapoptotic gene. Additionalanti-apoptotic genes that can be used in accordance with the presentinvention include, but are not limited to, E1B-9K, Aven, Mc1.

The disclosure also provides, in another embodiment, a cell culturecomposition comprises mammalian cells adapted to produce neublastinantibodies, or fragments thereof. Also provided are useful cells forthis purpose include, but are limited to, CHO cells, and retinal-derivedcells, and neublastin antibodies, or fragments thereof, made inmammalian cells.

The present invention provides a method of culturing cells, comprisingcontacting the cells with a medium disclosed herein.

Cell cultures can be cultured in a batch culture, fed batch culture or aperfusion culture. In one embodiment, a cell culture according to amethod of the present invention is a batch culture. In anotherembodiment, a cell culture according to a method of the presentinvention is a fed batch culture. In a further embodiment, a cellculture according to a method of the present invention is a perfusionculture.

In one embodiment, a cell culture according to a method of the presentinvention is a serum-free culture. In another embodiment, a cell cultureaccording to a method of the present invention is a chemically definedculture. In a further embodiment, a cell culture according to a methodof the present invention is an animal protein free culture.

In one embodiment, a cell culture is contacted with a medium describedherein during the growth phase of the culture. In another embodiment, acell culture is contacted with a medium described herein during theproduction phase of the culture.

In one embodiment, a cell culture according to the invention iscontacted with a feed medium described herein during the productionphase of the culture. In one embodiment, the culture is supplementedwith the feed medium between about 1 and about 25 times during thesecond time period. In another embodiment, a culture is supplementedwith the feed medium between about 1 and about 20 times, between about 1and about 15 times, or between about 1 and about 10 times during thefirst time period. In a further embodiment, a culture is supplementedwith the feed medium at least once, at least twice, at least threetimes, at least four times, at least five times, at least 6 times, atleast 7 times, at least 8 times, at least 9 times, at least 10 times, atleast 1 times, at least 12 times, at least 13 times, at least 14 times,at least 15 times, at least 20 times, at least 25 times. In a specificembodiment, the culture is a fed batch culture. In another specificembodiment, the culture is a perfusion culture.

A culture according to the invention can be contacted with a feed mediumdescribed herein at regular intervals. In one embodiment, the regularinterval is about once a day, about once every two days, about onceevery three days, about once every 4 days, or about once every 5 days.In a specific embodiment, the culture is a fed hatch culture. In anotherspecific embodiment, the culture is a perfusion culture.

A culture according to the invention can be contacted with a feed mediumdescribed herein on an as needed basis based on the metabolic status ofthe culture. In one embodiment, a metabolic marker of a fed hatchculture is measured prior to supplementing the culture with a feedmedium described herein. In one embodiment, the metabolic marker isselected from the group consisting of: lactate concentration, ammoniumconcentration, alanine concentration, glutamine concentration, glutamateconcentration, cell specific lactate production rate to the cellspecific glucose uptake rate ratio (LPR/GUR ratio), and Khodarnine 123specific cell fluorescence. In one embodiment, an LPR/GUR value of >0.1indicates the need to supplement the culture with a feed mediumdescribed herein. In a further specific embodiment, a lactateconcentration of >3 g/L indicates the need to supplement the culturewith a feed medium described herein. In another embodiment, a cultureaccording to the present invention is supplemented with a feed mediumdescribed herein when the LPR/GUR value of the culture is >0.1 or whenthe lactate concentration of the culture is >3 g/L. In a specificembodiment, the culture is a fed batch culture. In another specificembodiment, the culture is a perfusion culture.

In one embodiment, a medium described herein is a feed medium for a fedbatch cell culture. A skilled artisan understands that a fed batch cellculture can be contacted with a feed medium more than once. In oneembodiment, a fed batch cell culture is contacted with a mediumdescribed herein only once. In another embodiment, a fed batch cellculture is contacted with a medium described herein more than once, forexample, at least twice, at least three times, at least four times, atleast five times, at least six times, at least seven times, or at leastten times.

In accordance with the present invention, the total volume of feedmedium added to a cell culture should optimally be kept to a minimalamount. For example, the total volume of the feed medium added to thecell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45 or 50% of the volume of the cell culture prior to adding the feedmedium.

Cell cultures can be grown to achieve a particular cell density,depending on the needs of the practitioner and the requirement of thecells themselves, prior to being contacted with a medium describedherein. In one embodiment, the cell culture is contacted with a mediumdescribed herein at a viable cell density of 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent ofmaximal viable cell density. In a specific embodiment, the medium is afeed medium.

Cell cultures can be allowed to grow for a defined period of time beforethey are contacted with a medium described herein. In one embodiment,the cell culture is contacted with a medium described herein at day 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the cell culture. Inanother embodiment, the cell culture is contacted with a mediumdescribed herein at week 1, 2, 3, 4, 5, 6, 7, or 8 of the cell culture.In a specific embodiment, the medium is a feed medium.

Cell cultures can be cultured in the production phase for a definedperiod of time. In one embodiment, the cell culture is contacted with afeed medium described herein at day 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 of the production phase.

A culture according to the invention can be maintained in productionphase for between about 1 day and about 30 days. In one embodiment, aculture is maintained in production phase for between about 1 day andabout 30 days, between about 1 day and about 25 days, between about 1day and about 20 days, about 1 day and about 15 days, about 1 day andabout 14 days, about 1 day and about 13 days, about 1 day and about 12days, about 1 day and about 11 days, about 1 day and about 10 days,about 1 day and about 9 days, about 1 day and about 8 days, about 1 dayand about 7 days, about 1 day and about 6 days, about 1 day and about 5days, about 1 day and about 4 days, about 1 day and about 3 days, about2 days and about 25 days, about 3 days and about 25 days, about 4 daysand about 25 days, about 5 days and about 25 days, about 6 days andabout 25 days, about 7 days and about 25 days, about 8 days and about 25days, about 9 days and about 25 days, about 10 days and about 25 days,about 15 days and about 25 days, about 20 days and about 25 days, about2 days and about 30 days, about 3 days and about 30 days, about 4 daysand about 30 days, about 5 days and about 30 days, about 6 days andabout 30 days, about 7 days and about 30 days, about 8 days and about 30days, about 9 days and about 30 days, about 10 days and about 30 days,about 15 days and about 30 days, about 20 days and about 30 days, orabout 25 days and about 30 days. In another embodiment, a culture ismaintained in production phase for at least about 1 day, at least about2 days, at least about 3 days, at least about 4 days, at least about 5days, at least about 6 days, at least about 7 days, at least about 8days, at least about 9 days, at least about 10 days, at least about 11days, at least about 12 days, at least about 15 days, at least about 20days, at least about 25 days, or at least about 30 days. In a furtherembodiment, a culture is maintained in production phase for about 1 day,about 2 days, about 3 days, about 4 days, about 5 days, about 6 days,about 7 days, about 8 days, about 9 days, about 10 days, about 11 days,about 12 days, about 15 days, about 20 days, about 25 days, or about 30days.

The present invention further provides a method of preventing orreducing metabolic imbalance in a cell culture. Metabolic imbalance canbe monitored by measuring the levels of metabolites in the cell culture.For example, metabolic imbalance can be detected by monitoring lactateproduction, ammonium production, the ratio of cell specific lactateproduction rate (LPR) to cell specific glucose uptake rate (GUR),alanine consumption, or glutamine consumption in a cell culture. In oneembodiment, metabolic imbalance is signaled by increased lactateproduction, increased ammonium production or an increase in the cellspecific lactate production rate to cell specific glucose uptake rateratio (“LPR/GUR ratio”). In another embodiment, metabolic imbalance issignaled by an increase in alanine consumption or by an increase inglutamine consumption.

In one embodiment, a method of culturing cells according to the presentinvention prevents or reduces mitochondria dysfunction or metabolicimbalance during the exponential growth phase. In another embodiment, amethod of culturing cells according to the present invention prevents orreduces mitochondria dysfunction or metabolic imbalance after the cellspassed the exponential growth phase. In a further embodiment, a methodof culturing cells—according to the present invention prevents orreduces mitochondrial dysfunction or metabolic imbalance during theproduction phase.

The present invention also provides a method of decreasing lactateproduction by a cell culture, comprising contacting the cell culturewith a medium described herein. In one embodiment, the lactateproduction of cells maintained in a culture medium described herein isbetween about 5% and about 90%, between about S %, and about 80%,between about 5%, and about 70%, between about 5%, and about 50%,between about 5%, and about 40%, between about 5%, and about 30%,between about 5%, and about 20%, between about 10%, and about 90%,between about 20%, and about 90%, between about 30%, and about 90%, orbetween about 50%, and about 90%, or at least about 5%, at least about10%, at least about 20%, at least about 30%, at least about 50%, or atleast about 90%, or about 5%, about 10%, about 20% about 30%, about 50%,or about 90% lower than the lactate production of cells maintained in aculture medium that is substantially free from dextran sulfate or ferriccitrate. In one embodiment, a cell culture described herein comprisesbetween about 0.1 g/L and about 6 g/L, between about 0.1 g/L and about 5g/L, between about 0.1 g/L and about 4 g/L, or between about 0.1 g/L andabout 3 g/L of lactate. In another embodiment, a cell culture describedherein comprises less than about 6 WI, about 5 g/L, about 4 g/L, about 3g/L, about 2 g/L or about 1 g/L lactate.

The present invention also provides a method of decreasing ammoniumproduction by a cell culture, comprising contacting the cell culturewith a medium described herein. In one embodiment, the ammoniumproduction of cells maintained in a culture medium described herein isbetween about 5% and about 90%, between about 5%, and about 80%, betweenabout 5%, and about 70%, between about 5%, and about 50%, between about5%, and about 40%, between about 5%, and about 30%, between about 5%,and about 20%, between about 10%, and about 90%, between about 20%, andabout 90%, between about 30%, and about 90%, or between about 50%, andabout 90%, or at least about 5%, at least about 10%, at least about 20%,at least about 30%, at least about 50%, or at least about 90%, or about5%, about 10%, about 20%, about 30%, about 50%, or about 90% lower thanthe ammonium production of cells maintained in a culture medium that issubstantially free from dextran sulfate or ferric citrate. In oneembodiment, a cell culture described herein comprises between about 0.1mM and about 20 mM, about 0.1 mM and about 15 mM, about 0.1 mM and about14 mM, about 0.1 mM and about 13 mM, about 0.1 mM and about 12 mM, about0.1 mM and about 11 mM, about 0.1 mM and about 10 mM, about 0.1 mM andabout 9 mM, about 0.1 mM and about 8 mM, about 0.1 mM and about 7 mM,about 0.1 mM and about 6 mM, about 0.1 mM and about 5 mM, about 0.1 mMand about 4 mM, about 0.1 mM and about 3 mM, about 0.1 mM and about 2mM, about 0.1 mM and about 1 mM, about 0.5 mM and about 20 mM, about 0.5mM and about 15 mM, about 0.5 mM and about 14 mM, about 0.5 mM and about13 mM, about 0.5 mM and about 12 mM, about 0.5 mM and about 11 mM, about0.5 mM and about 10 mM, about 0.5 mM and about 9 mM, about 0.5 mM andabout 8 mM, about 0.5 mM and about 7 mM, about 0.5 mM and about 6 mM,about 0.5 mM and about 5 mM, about 0.5 mM and about 4 mM, about 0.5 mMand about 3 mM, about 0.5 mM and about 2 mM, about 0.5 mM and about 1mM, about 1 mM and about 20 mM, about 1 mM and about 15 mM, about 1 mMand about 14 mM, about 1 mM and about 13 mM, about 1 mM and about 12 mM,about 1 mM and about 11 mM, about 1 mM and about 10 mM, about 1 mM andabout 9 mM, about 1 mM and about 8 mM, about 1 mM and about 7 mM about 1mM and about 6 mM, about 1 mM and about 5 mM, about 1 mM and about 4 mM,about 1 mM and about 3 mM, or about 1 mM and about 2 mM ammonium. Inanother embodiment, a cell culture described herein comprises less thanabout 20 mM, about 19 mM, about 18 mM, about 17 mM, about 16 mM, about15 mM, about 14 mM; about 13 mM, about 12 mM, about 11 mM, about 10 mM,about 9 mM, about 8 mM, about 7 mM, about 6 mM, about 5 mM, about 4 mM,about 3 mM, about 2 mM, about 1 mM, or about 0.5 mM ammonium.

The present invention further provides a method of producing a proteinor polypeptide of interest, comprising culturing cells capable ofproducing the protein or polypeptide of interest in a culture comprisinga medium described herein; and isolating the protein or polypeptide fromthe culture. In one embodiment, the protein or polypeptide of interestis a recombinant protein or polypeptide. In one embodiment, the proteinor polypeptide of interest is an enzyme, receptor, antibody, hormone,regulatory factor, antigen, or binding agent. In a specific embodiment,the protein is an antibody, which can be, but is not limited to, aneublastin antibody, such as a monoclonal antibody.

In one embodiment of the present invention, a cell culture comprising amedium described herein can be maintained in production phase longerthan a cell culture that does not comprise exogenous dextran sulfate. Askilled artisan readily understands that an extended production phasecan lead to an increase in the total amount of polypeptide produce bythe cell culture. In one embodiment, a method of producing a polypeptideof interest according to the present invention produces more polypeptidethan the amount produced by a method that does not comprise maintainingcells capable of producing the polypeptide in a culture comprisingexogenous dextran sulfate. In one embodiment, a method according to thepresent invention produces between about 5% and about 500%, about 5% andabout 250%, about 5% and about 100%, about 5% and about 80%, about 5%and about 50%, about 5% and about 30%, about 10% and about 500%, about20% and about 500%, about 30% and about 500%, about 50% and about 500%,or about 100% and about 500% more protein or polypeptide. In anotherembodiment, a method according to the present invention produces atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 40%, atleast about 50%, at least about 70%, at least about 90%, or at leastabout 100% more protein or polypeptide. In another embodiment, a methodaccording to the present invention produces at least about 2 times,three times, four times, five times or ten times more protein orpolypeptide. In a specific embodiment, the protein or polypeptide is anantibody.

In one embodiment, a method of producing a polypeptide of interestaccording to the present invention produces a higher titer of thepolypeptide in the cell culture than the titer produced by a method thatdoes not comprise maintaining the cells in a culture comprising dextransulfate. In one embodiment, a method according to the present inventionproduces between about 5% and about 500%, about 5% and about 250%, about5% and about 100%, about 5% and about 80%, about 5% and about 50%, about5% and about 30%, about 10% and about 500%, about 20% and about 500%,about 30% and about 500%, about 50% and about 500%, or about 100% andabout 500% higher titer. In another embodiment, a method according tothe present invention produces at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 40%, at least about 50%, at least about 70%, atleast about 90%, or at least about 100% higher titer. In anotherembodiment, a method according to the present invention produces atleast about 2 times, three times, four times, five times or ten timeshigher titer.

In a specific embodiment, the protein or polypeptide, is an antibody.

In a specific embodiment, a method of producing a polypeptide ofinterest according to the present invention produces a maximum proteinor polypeptide titer of at least about 2 g/liter, at least about 2.5g/liter, at least about 3 g/liter, at least about 3.5 g/liter, at leastabout 4 g/liter, at least about 4.5 g/liter, at least about 5 g/liter,at least about 6 g/liter, at least about 7 g/liter, at least about 3g/liter, at least about 9 g/liter, or at least about 10 g/liter. Inanother embodiment, the method according to the present inventionproduces a maximum protein or polypeptide titer of between about 1g/liter and about 10 g/liter, about 1.5 g/liter and about 10 g/liter,about 2 g/liter and about 10 g/liter, about 2.5 g/liter and about 10g/liter, about 3 g/liter and about 10 g/liter, about 4 g/liter and about10 g/liter, about 5 g/liter and about 10 g/liter, about 1 g/liter andabout 5 g/liter, about 1 g/liter and about 4.5 g/liter, or about 1g/liter and about 4 g/liter. In a specific embodiment, the protein orpolypeptide is an antibody. In another embodiment, the protein orpolypeptide is a blood clotting factor.

The invention further provides a conditioned cell culture mediumproduced by a method described herein.

In one embodiment, a conditioned cell culture medium according to theinvention comprises a recombinant protein or polypeptide. In a specificembodiment, a conditioned cell culture medium according to the inventioncomprises a recombinant protein or polypeptide, at a titer of at leastabout 2 g/liter, at least about 2.5 g/liter, at least about 3 g/liter,at least about 3.5 g/liter, at least about 4 g/liter, at least about 4.5g/liter at least about 5 g/liter, at least about 6 g/liter, at leastabout 7 g/liter, at least about 8 g/liter, at least about 9 g/liter, orat least about 10 g/liter, or a liter of between about 1 g/liter andabout 10 g/liter, about 1.5 g/liter and about 10 g/liter, about 2g/liter and about 10 g/liter, about 2.5 g/liter and about 10 g/liter,about 3 g/liter and about 10 g/liter, about 4 g/liter and about 10g/liter, about 5 g/liter and about 10 g/liter, about 1 g/liter and about5 g/liter, about 1 g/liter and about 4.5 g/liter, or about 1 g/liter andabout 4 g/liter. In another embodiment, a conditioned cell culturemedium according to the invention comprises recombinant protein orpolypeptide at a higher titer than the titer obtained without the use ofa medium described herein. In a specific embodiment, the protein orpolypeptide is an antibody.

Polypeptides

Any polypeptide that is expressible in a host cell can be produced inaccordance with the present invention. The polypeptide can be expressedfrom a gene that is endogenous to the host cell, or from a gene that isintroduced into the host cell through genetic engineering. Thepolypeptide can be one that occurs in nature, or can alternatively havea sequence that was engineered or selected by the hand of man. Anengineered polypeptide can be assembled from other polypeptide segmentsthat individually occur in nature, or can include one or more segmentsthat are not naturally occurring.

Polypeptides that can desirably be expressed in accordance with thepresent invention will often be selected on the basis of an interestingbiological or chemical activity, for example, the present invention canbe employed to express any pharmaceutically or commercially relevantenzyme, receptor, antibody, hormone, regulatory factor, antigen, bindingagent, etc.

Particularly useful polypeptides are those that are highly negativelycharged. Examples of highly negatively charged polypeptides include, butare not limited to, neublastin and Factor VIII.

Antibodies

Given the large number of antibodies currently in use or underinvestigation as pharmaceutical or other commercial agents, productionof antibodies is of particular interest in accordance with the presentinvention. Antibodies are proteins that have the ability to specificallybind a particular antigen. Any antibody that can be expressed in a hostcell can be used in accordance with the present invention. In oneembodiment, the antibody to be expressed is a monoclonal antibody.

Particular antibodies can be made, for example, by preparing andexpressing synthetic genes that encode the recited amino acid sequencesor by mutating human germline genes to provide a gene that encodes therecited amino acid sequences. Moreover, these antibodies can heproduced, e.g., using one or more of the following methods.

Numerous methods are available for obtaining antibodies, particularlyhuman antibodies. One exemplary method includes screening proteinexpression libraries, e.g. phage or ribosome display libraries. Phagedisplay is described, for example, U.S. Pat. No. 5,223,409; Smith (1985)Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; and WO90/02809. The display of Fab's on phage is described, e.g., in U.S. Pat.Nos. 5,658,727; 5,667,988; and 5,885,793.

In addition to the use of display libraries, other methods can be usedto obtain an antibody. For example, a protein or a peptide thereof canbe used as an antigen in a non-human animal, e.g., a rodent, e.g., amouse, hamster, or rat.

In one embodiment, the non-human animal includes at least a part of ahuman immunoglobulin gene. For example, it is possible to engineer mousestrains deficient in mouse antibody production with large fragments ofthe human Ig loci. Using the hybridoma technology, antigen-specificmonoclonal antibodies derived from the genes with the desiredspecificity can be produced and selected. See, e.g., XENOMOUSE™, Greenet al. (1994) Nature Genetics 7:13-21, U.S. 2003-0070185, WO 96/34096,and WO 96/33735.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified, e.g., humanized or deimmunized.Winter describes an exemplary CDR grafting method that can be used toprepare humanized antibodies described herein (U.S. Pat. No. 5,225,539).All or some of the CDRs of a particular human antibody can be replacedwith at least a portion of a non-human antibody. In one embodiment, itis only necessary to replace the CDRs required for binding or bindingdeterminants of such CDRs to arrive at a useful humanized antibody thatbinds to an antigen.

Humanized antibodies can be generated by replacing sequences of the Fvvariable region that are not directly involved in antigen binding withequivalent sequences from human Fv variable regions, General methods forgenerating humanized antibodies are provided by Morrison, S. L. (1985)Science 229:1202-1207, by Oi et al. (1986) BioTechniques 4:214, and byU.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762; 5,859,205; and6,407,213. Those methods include isolating, manipulating, and expressingthe nucleic-acid sequences that encode all or part of immunoglobulin Fvvariable regions from at least one of a heavy or light chain. Sources ofsuch nucleic acid are well known to those skilled in the art and, forexample, can be obtained from a hybridoma producing an antibody againsta predetermined target, as described above, from germline immunoglobulingenes, or from synthetic constructs. The recombinant DNA encoding thehumanized antibody can then be cloned into an appropriate expressionvector. In one embodiment, the expression vector comprises apolynucleotide encoding a glutamine synthetase polypeptide. (See, e.g.,Porter et al., Biotechnol Prog 26(5):1446-54 (2010).

The antibody can include a human Fc region, e.g., a wild-type Fc regionor an Fc region that includes one or more alterations. In oneembodiment, the constant region is altered, e.g., mutated, to modify theproperties of the antibody (e.g., to increase or decrease one or moreof: Fc receptor binding, antibody glycosylation, the number of cysteineresidues, effector cell function, or complement function). For example,the human IgG constant region can be mutated at one or more residues,e.g., one or more of residues 234 and 237. Antibodies can have mutationsin the CH2 region of the heavy chain that reduce or alter effectorfunction, e.g., Fc receptor binding and complement activation. Forexample, antibodies can have mutations such as those described in U.S.Pat. Nos. 5,624,821 and 5,648,260. Antibodies can also have mutationsthat stabilize the disulfide bond between the two heavy chains of animmunoglobulin, such as mutations in the hinge region of IgG14, asdisclosed in the art (e.g., Angal et. al, (1993) Mol. Immunol.30:105-08). See also, e.g., U.S. 2005-0037000.

In other embodiments, the antibody can be modified to have an alteredglycosylation pattern (i.e., altered from the original or nativeglycosylation pattern). As used in this context, “altered” means havingone or more carbohydrate moieties deleted, and/or having one or moreglycosylation sites added to the original antibody. Addition ofglycosylation sites to the presently disclosed antibodies can beaccomplished by altering the amino acid sequence to containglycosylation site consensus sequences; such techniques are well knownin the art. Another means of increasing the number of carbohydratemoieties on the antibodies is by chemical or enzymatic coupling ofglycosides to the amino acid residues of the antibody. These methods aredescribed in, e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit,Rev. Biochem. 22:259-306. Removal of any carbohydrate moieties presenton the antibodies can be accomplished chemically or enzymatically asdescribed in the art (Hakimuddin et al, (1987) Arch. Biochem. Biophys.259:52; Edge et al. (1981) Anal. Biochem. 118:131; and Thotakura et al.(1987) Meth. Enzymol, 138:350), See, e.g., U.S. Pat. No. 5,869,046 for amodification that increases in vivo half-life by providing a salvagereceptor binding epitope.

The antibodies can be in the form of full length antibodies, or in theform of fragments of antibodies, e.g., Fab, F(ab′)2, Fd, dAb, and scFvfragments. Additional forms include a protein that includes a singlevariable domain, e.g., a camelid or camelized domain. See, e.g., U.S.2005-0079574 and Davies et al, (1996) Protein Eng. 9(6):531-7.

In one embodiment, the antibody is an antigen-binding fragment of a fulllength antibody, e.g., a Fab, F(ab′)2, Fv, or a single chain Fvfragment. Typically, the antibody is a full length antibody. Theantibody can be a monoclonal antibody or a mono specific antibody.

In another embodiment, the antibody can be a human, humanized,CDR-grafted, chimeric, mutated, affinity matured, deimmunized, syntheticor otherwise in vitro-generated antibody, and combinations thereof.

The heavy and light chains of the antibody can be substantiallyfull-length. The protein can include at least one, and preferably two,complete heavy chains, and at least one, and preferably two, completelight chains) or can include an antigen-binding fragment (e.g., a Fab,F(ab′)2, Fv or a single chain Fv fragment). In yet other embodiments,the antibody has a heavy chain constant region chosen from, e.g., IgG1,IgG2, IgG13, IgG4, IgM, IgGA1, IgA2, IgD, and IgE; particularly, chosenfrom, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g.,IgG1). Typically, the heavy chain constant region is human or a modifiedform of a human constant region. In another embodiment, the antibody hasa light chain constant region chosen from, e.g., kappa or lambda,particularly, kappa (e.g., human kappa).

Receptors

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes receptors. Receptorsare typically trans-membrane glycoproteins that function by recognizingan extra-cellular signaling ligand. Receptors typically have a proteinkinase domain in addition to the ligand recognizing domain, whichinitiates a signaling pathway by phosphorylating target intracellularmolecules upon binding the ligand, leading to developmental or metabolicchanges within the cell. In one embodiment, the receptors of interestare modified so as to remove the transmembrane and/or intracellulardomain(s), in place of which there can optionally be attached anIg-domain. In one embodiment, receptors to be produced in accordancewith the present invention are receptor tyrosine kinases (RTKs). The RTKfamily includes receptors that are crucial for a variety of functionsnumerous cell types (see, e.g., Yarden and Ullrich, Ann. Rev. Biochem.57:433-478, 1988; Ullrich and Schlessinger (1990) Cell 61:243,254).Non-limiting examples of RTKs include members of the fibroblast growthfactor (FGF) receptor family, members of the epidermal growth factorreceptor (EGF) family, platelet derived growth factor (PDGF) receptor,tyrosine kinase with immunoglobulin and EGF homology domains-1 (TIE-1)and TIE-2 receptors (Sato et al., Nature 376(6535):70-74 (1995),incorporated herein by reference) and c-Met receptor, some of which havebeen suggested to promote angiogenesis, directly or indirectly (Mustonenand Alitalo, J. Cell Biol. 129:895-898, 1995). Other non-limitingexamples of RTK's include fetal liver kinase 1 (FLK-1) (sometimesreferred to as kinase insert domain-containing receptor (KDR) (Terman etal., Oncogene 6:1677-83, 1991) or vascular endothelial cell growthfactor receptor 2 (VEGFR-2)), fins-like tyrosine kinase-1 (Flt-1)(DeVries et al, Science 255:989-991, 1992; Shibuya et al., Oncogene5:519-524, 1990), sometimes referred to as vascular endothelial cellgrowth factor receptor 1 (VEGFR-1), neuropilin-1, endoglin, endosialin,and Axl. Those of ordinary skill in the art will be aware of otherreceptors that can be expressed in accordance with the presentinvention.

Growth Factors and Other Signaling Molecules

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes growth factors andother signaling molecules. Growth factors are typically glycoproteinsthat are secreted by cells and bind to and activate receptors on othercells, initiating a metabolic or developmental change in the receptorcell.

Non-limiting examples of mammalian growth factors and other signalingmolecules include cytokines; epidermal growth factor (EGF);platelet-derived growth factor (PDGF); fibroblast growth factors (FGFs)such as aFGF and bFGF; transforming growth factors (TGFs) such asTGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3,TGF-beta 4, or TGF-beta 5; insulin-like growth factor-I, and -II (IGF-Iand IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factorbinding proteins; CD proteins such as CD-3., CD-4, CD-8, and CD-19;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such as interferon-alpha,-beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF,GM-CSF, and G-CSF; interleukins (TLs), e.g., IL-1 to IL-10; tumornecrosis factor (TNF) alpha and beta; insulin A-chain; insulin B-chain;proinsulin; follicle stimulating hormone; calcitonin; luteinizinghormone; glucagon; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin, hemopoietic growth factor; enkephalinase; RANTES(regulated on activation normally T-cell expressed and secreted); humanmacrophage inflammatory protein (MIP-1-alpha); mullerian-inhibitingsubstance; relaxin A-chain; relaxin B-chain; prorelaxin; mousegonadotropin-associated peptide; neurotrophic factors such asbone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6(NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-beta,One of ordinary skill in the art will be aware of other growth factorsor signaling molecules that can be expressed in accordance with thepresent invention.

Clotting Factors

In some embodiments, the protein of interest comprises a clottingfactor. “Clotting factor”, as used herein, means any molecule, or analogthereof, which prevents or decreases the duration of a bleeding episodein a subject with a hemostatic disorder. For example, a clotting factorfor the invention can be a full-length clotting factor, a matureclotting factor, or a chimeric clotting factor. In other words, it meansany molecule having clotting activity. Clotting activity, as usedherein, means the ability to participate in a cascade of biochemicalreactions that culminates in the formation of a fibrin clot and/orreduces the severity, duration or frequency of hemorrhage or bleedingepisode. Examples of clotting factors can be found in U.S. Pat. No.7,404,956.

In one embodiment, the clotting factor is Factor VIII, Factor IX, FactorXI, Factor XII, fibrinogen, prothrombin, Factor V, Factor VII, Factor X,Factor XIII or von Willebrand Factor. The clotting factor can be afactor that participates in the extrinsic pathway. The clotting factorcan be a factor that participates in the intrinsic pathway.Alternatively, the clotting factor can be a factor that participates inboth the extrinsic and intrinsic pathway.

In one embodiment, the clotting factor can be a non-human clottingfactor or a non-human clotting factor, e.g., derived from a non-humanprimate, a pig or any mammal. The clotting factor can be chimericclotting factor, e.g., the clotting factor can comprise a portion of ahuman clotting factor and a portion of a porcine clotting factor or aportion of a first non-human clotting factor and a portion of a secondnon-human clotting factor.

In another embodiment, the clotting factor can be an activated clottingfactor. Alternatively, the clotting factor can be an inactive form of aclotting factor, e.g., a zymogen. The inactive clotting factor canundergo activation subsequent to being linked to at least a portion ofan immunoglobulin constant region. The inactive clotting factor can beactivated subsequent to administration to a subject. Alternatively, theinactive clotting factor can be activated prior to administration.

In certain embodiments, the clotting factor is a Factor VIII protein.“Factor VIII protein” or “FVIII protein” as used herein, meansfunctional Factor VIII protein in its normal role in coagulation, unlessotherwise specified. Thus, the term FVIII includes variant proteins thatare functional. In one embodiment, the FVIII protein is the human,porcine, canine, rat, or marine FVIII protein. A functional FVIIIprotein can be a fusion protein, such as, but not limited to, a fusionprotein comprising a fully or partially B-domain deleted FVIII, at leasta portion of an immunoglobulin constant region, e.g., an Fc domain, orboth. Myriad functional FVIII variants have been constructed and can beused as recombinant FVIII proteins as described herein, See PCTPublication Nos. WO 2011/069164 A2, WO 2012/006623 A2, WO 2012/006635A2, or WO 2012/006633 A2, all of which are incorporated herein byreference in their entireties.

A great many functional FVIII variants are known. In addition, hundredsof nonfunctional mutations in FVIII have been identified in hemophiliapatients. See, e.g., Cutler et al., Hum. Mutat. 19:274-8 (2002),incorporated herein by reference in its entirety. In addition,comparisons between FVIII from humans and other species have identifiedconserved residues that are likely to be required for function. See,e.g., Cameron et al., Thromb. Haernost. 79:317-22 (1998) and U.S. Pat.No. 6,251,632, incorporated herein by reference in their entireties.

In certain aspects, a recombinant FVIII protein of the invention ischimeric. A “chimeric protein,” or “chimeric polypeptide” as usedherein, means a protein or polypeptide that includes within it at leasttwo stretches of amino acids from different sources, e.g., a FVIIIprotein comprising a heterologous moiety. In one embodiment, theheterologous moiety can be a half-life extending moiety. Examples of theheterologous moieties include, but are not limited to, an immunoglobulinconstant region or a fragment thereof, e.g., an Fc region or an FeRnbinding partner, a VWF molecule, or a fragment thereof, albumin, albuminbinding polypeptide, Fc, PHS, the β subunit of the C-terminal peptide(CTP) of human chorionic gonadotropin, polyethylene glycol (PEG),hydroxyethyl starch (HES), albumin-binding small molecules, orcombinations thereof. In some embodiments, the chimeric protein is aFVIII monomer dimer hybrid.

A long-acting or long-lasting FIX polypeptide useful for the inventionis a chimeric polypeptide comprising a FIX polypeptide and an FcRnbinding partner. The FIX polypeptide of the invention comprises afunctional Factor IX polypeptide in its normal role in coagulation,unless otherwise specified. Thus, the FIX polypeptide includes variantpolypeptides that are functional and the polynucleotides that encodesuch functional variant polypeptides. In one embodiment, the FIXpolypeptides are the human, bovine, porcine, canine, feline, and murineFIX polypeptides. The full length polypeptide and polynucleotidesequences of FIX are known, as are many functional variants, e.g.,fragments, mutants and modified versions. FIX polypeptides includefull-length FIX, full-length FIX minus Met at the N-terminus,full-length FIX minus the signal sequence, mature, FIX (minus the signalsequence and propeptide), and mature FIX with an additional Met at theN-terminus. FIX can be made by recombinant means (“recombinant FactorIX” or “rFIX”), i.e., it is not naturally occurring or derived fromplasma.

The clotting factor can also include a FIX protein or any variant,analog, or functional fragments thereof. A great many functional FIXvariants are known. International publication number WO 02/040544 A3,which is herein incorporated by reference in its entirety, disclosesmutants that exhibit increased resistance to inhibition by heparin atpage 4, lines 9-30 and page 15, lines 6-31. International publicationnumber WO 03/020764 A2, which is herein incorporated by reference in itsentirety, discloses FIX mutants with reduced T cell immunogenicity inTables 2 and 3 (on pages 14-24), and at page 12, lines 1-27.International publication number WO 2007/149406 A2, which is hereinincorporated by reference in its entirety, discloses functional mutantFIX molecules that exhibit increased protein stability, increased invivo and in vitro half-life, and increased resistance to proteases atpage 4, line 1 to page 19, line 11. WO 2007/149406 A2 also discloseschimeric and other variant FIX molecules at page 19, line 12 to page 20,line 9. International Pub. no WO 08/118507 A2, which is hereinincorporated by reference in its entirety, discloses FIX mutants thatexhibit increased clotting activity at page 5, line 14 to page 6, line5. International Pub. no WO 09/051717 A2, discloses FIX mutants havingan increased number of N.-linked and/or O-linked glycosylation sites,which results in an increased half-life and/or recovery at page 9, line11 to page 20, line 2. International publication number WO 09/137254 A2,which is herein incorporated by reference in its entirety, alsodiscloses Factor IX mutants with increased numbers of glycosylationsites at page 2, paragraph [006] to page 5, paragraph [011] and page 16,paragraph [044] to page 24, paragraph [057]. International Pub. no WO09/130198 A2, which is herein incorporated by reference in its entirety,discloses functional mutant FIX molecules that have an increased numberof glycosylation sites, which result in an increased half-life, at page4, line 26 to page 12, line 6. International Pub. no WO 09/140015 A2,which is herein incorporated by reference in its entirety, disclosesfunctional FIX mutants that an increased number of Cys residues, whichmay be used for polymer (e.g., PEG) conjugation, at page 11, paragraph[0043] to page 13, paragraph [0053]. The FIX polypeptides described inInternational Application No. PCT/US2011/043569 filed Jul. 11, 2011 andpublished as WO 2012/006624 on Jan. 12, 2012 are also incorporatedherein by reference in its entirety.

In addition, hundreds of non-functional mutations in FIX have beenidentified in hemophilia patients, many of which are disclosed in Table1, at pages 11-14 of International Pub. no WO 09/137254 A2, which isherein incorporated by reference in its entirety. Such non-functionalmutations are not included in the invention, but provide additionalguidance for which mutations are more or less likely to result in afunctional FIX polypeptide.

In some embodiments, the chimeric protein of the invention is a FIXmonomerdimer hybrid. Monomer-dimer hybrid can comprise two polypeptidechains, one chain comprising a FIX polypeptide and a first Fc region,and another chain comprising, consisting essentially of, or consistingof a second Fc region. In certain aspects, a FIX monomer dimer hybridconsists essentially of or consists of two polypeptide chains, a firstchain consisting essentially of or consisting of a FIX polypeptide and asecond chain consisting essentially of or consisting of a second Fcregion.

In some embodiments, a clotting factor is a mature form of Factor VII ora variant thereof. Factor VII (F-VII, F7; also referred to as Factor 7,coagulation factor VII, serum factor VII, serum prothrombin conversionaccelerator, SPCA, proconvertin and eptacog alpha) is a serine proteasethat is part of the coagulation cascade. FVII includes a Gla domain, twoEGF domains (EGF-1 and EGF-2), and a serine protease domain (orpeptidase S1 domain) that is highly conserved among all members of thepeptidase S1 family of serine proteases, such as for example withchymotrypsin. FVII occurs as a single chain zymogen, an activatedzymogen-like two-chain polypeptide and a fully activated two-chain form.

Exemplary FVII variants include those with increased specific activity,e.g., mutations that increase the activity of FVII by increasing itsenzymatic activity (Kcat or Km). Such variants have been described inthe art and include, e.g., mutant forms of the molecule as described forexample in Persson et al., 2001, PNAS 98:13583; Petrovan and Ruf, 2001.J. Biol, Chem. 276:6616; Persson et al., 2001 J. Biol. Chem. 276:29195;Soejima et al., 2001, J. Biol. Chem. 276:17229; Soejima et al., 2002, J.Biol. Chem. 247:49027.

In one embodiment, a variant form of FV1I includes the mutations.Exemplary mutations include V158D-E296V-M298Q. In another embodiment, avariant form of -FVII includes a replacement of amino acids 608-619(LQQSRKVGDSPN, corresponding to the 170-loop) from the FVII maturesequence with amino acids EASYPGK from the 170-loop of trypsin, Highspecific activity variants of FIX are also known-in the art. Forexample, Simioni et al., 2009 N.E. J. Med. 361:1671) describe an R338L,mutation. Chang et al. (1988 JBC 273:12089) and Pierri et al., (20.09Human Gene Therapy 20:479) describe an R338A mutation. Other mutationsare known in the art and include those described, e.g., in Zogg andBrandstetter. 2009 Structure 17:1669; Sichler et al., 2003, J. Biol.Chem. 278:4121; and Sturzebecher et al., 1997, FEBS Lett 412:295. Thecontents of these references are incorporated herein by reference.

Full activation, which occurs upon conformational change from azymogen-like form, occurs upon binding to is co-factor tissue factor.Also, mutations can be introduced that result in the conformation changein the absence of tissue factor. Hence, reference to FVIIa includes bothtwo-chain forms thereof: the zymogen-like form (e.g., activatable FVII),and the fully activated two-chain form.

Various patents or applications disclosing examples of the clottingfactors useful for the invention are incorporated herein by reference.For example, various monomer dimer hybrid constructs comprising clottingfactors (FVII, FIX, and FVIII) are disclosed in U.S. Pat. Nos.7,404,945, 7,348,004, 7,862,820, 8,329,182, and 7,820,162, incorporatedherein by reference in their entireties, Examples of FVIII chimericprotein are additionally disclosed in US Appl. Nos. 61/734,954 or61/670,553, incorporated by reference in its entirety. Examples of FVIIchimeric protein are disclosed in U.S. Appln. No. 61/657,688.

G-Protein Coupled Receptors

Another class of polypeptides that have been shown to be effective aspharmaceutical and/or commercial agents includes growth factors andother signaling molecules. G-protein coupled receptors (GPCRs) areproteins that have seven transmembrane domains. Upon binding of a ligandto a GPCR, a signal is transduced within the cell which results in achange in a biological or physiological property of the cell.

GPCRs, along with G.-proteins and effectors (intracellular enzymes andchannels which are modulated by G-proteins), are the components of amodular signaling system that connects the state of intracellular secondmessengers to extracellular inputs. These genes and gene-products arepotential causative agents of disease.

The GPCR protein superfamily now contains over 250 types of paralogues,receptors that represent variants generated by gene duplications (orother processes), as opposed to orthologues, the same receptor fromdifferent species, The superfamily can be broken down into fivefamilies; Family I, receptors typified by rhodopsin and thebeta2-adrenergic receptor and currently represented by over 200 uniquemembers; Family II, the recently characterized parathyroidhormone/calcitonin/secretin receptor family; Family III, themetabotropic glutamate receptor family in mammals; Family IV, the cANIPreceptor family, important in the chemotaxis and development of D.discoideum; and Family V, the fungal mating pheromone receptors such asSTE2.

Viruses

Additionally, the present invention also provides methods for theproduction of viruses using a cell culture according to methods known tothose of skill in the field of virology. The viruses to be produced inaccordance with the present invention can be chosen from the range ofviruses known to infect the cultured, cell type. For instance, whenutilizing a mammalian cell culture, viruses can be chosen from thegenera of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses,flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses andadenoviruses. The virus used can be a wild-type virus, an attenuatedvirus, a reassortant virus, or a recombinant virus. In addition, insteadof actual virions being used to infect the cells with a virus, aninfectious nucleic acid clone can be utilized according to infectiousclone transfection methods known to those of skill in the field ofvirology. In one embodiment, the virus produced is an influenza virus.

Cells

Any eukaryotic cell or cell type susceptible to cell culture can beutilized in accordance with the present invention. For example, plantcells, yeast cells, animal cells, insect cells, avian cells or mammaliancells can be utilized in accordance with the present invention. In oneembodiment, the eukaryotic cells are capable of expressing a recombinantprotein or are capable of producing a recombinant or reassortant virus.

Non-limiting examples of mammalian cells that can be used in accordancewith the present invention include BALB/c mouse myeloma line (NSO/1,ECACC No: 85110503); human retinoblasts (PER,C6 (CruCell, Leiden, TheNetherlands)); monkey kidney CVI line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen. Virol., 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells ±DHFR (CHO, Urlaub and Chasin, Proc., Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinomacells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). In one embodiment, the present invention is used in theculturing of and expression of polypeptides from CHO cell lines. In aspecific embodiment, the CHO cell line is the DG44 CHO cell line. In aspecific embodiment, the CHO cell line comprises a vector comprising apolynucleotide encoding a glutamine synthetase polypeptide. In a furtherspecific embodiment, the CHO cell line expresses an exogenous glutaminesynthetase gene. (See, e.g., Porter et al., Biotechnol. Prog.26(5):1446-54 (2010)). In yet other embodiments, the CHO cell linecomprises a vector comprising a polynucleotide encoding a neublastinantibody or fragment thereof.

Additionally, any number of commercially and non-commercially availablehybridoma cell lines that express polypeptides or proteins can beutilized in accordance with the present invention. One skilled in theart will appreciate that hybridoma cell lines might have differentnutrition requirements and/or might require different culture conditionsfor optimal growth and polypeptide or protein expression, and will beable to modify conditions as needed,

The eukaryotic cells according to the present invention can be selectedor engineered to produce high levels of protein or polypeptide, or toproduce large quantities of virus. Often, cells are geneticallyengineered to produce high levels of protein, for example byintroduction of a gene encoding the protein or polypeptide of interestand/or by introduction of control elements that regulate expression ofthe gene (whether endogenous or introduced) encoding the polypeptide ofinterest.

The eukaryotic cells eau also be selected or engineered to survive inculture for extended periods of time. For example, the cells can begenetically engineered to express a polypeptide or polypeptides thatconfer extended survival on the cells. In one embodiment, the eukaryoticcells comprise a transgene encoding the Bcl-2 polypeptide or a variantthereof. See, e.g., U.S. Pat. No. 7,785,880. In a specific embodiment,the cells comprise a polynucleotide encoding the bol-xL polypeptide.See, e.g., Chiang G G, Sisk W P. 2005. Biotechnol. Bioeng.91(7):779-792.

The eukaryotic cells can also be selected or engineered to modify itsposttranslational modification pathways. In one embodiment, the cellsare selected or engineered to modify a protein glycosylation pathway. Ina specific embodiment, the cells are selected or engineered to expressan aglycosylated protein, e.g., an aglycosylated recombinant antibody.In another specific embodiment, the cells are selected or engineered toexpress an afucosylated protein, e.g., an afucosylated recombinantantibody.

The eukaryotic cells can also be selected or engineered to allowculturing in serum free medium.

Media

The cell culture of the present invention is prepared in any mediumsuitable for the particular cell being cultured. In some embodiments,the medium contains e.g., inorganic salts, carbohydrates (e.g., sugarssuch as glucose, galactose, maltose or fructose), amino acids, vitamins(e.g., B group vitamins (e.g., B12), vitamin A vitamin E, riboflavin,thiamine and biotin); fatty acids and lipids (e.g., cholesterol andsteroids), proteins and peptides (e.g., albumin, transferrin,fibronectin and fetuin), serum (e.g., compositions comprising albumins,growth factors and growth inhibitors, such as, fetal bovine serum,newborn calf serum and horse serum), trace elements (e.g., zinc, copper,selenium and tricarboxylic acid intermediates), hydrolysates (hydrolyzedproteins derived from plant or animal sources), and combinationsthereof. Commercially available media such as Ham's F10 (Sigma), MinimalEssential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium ([DMEM], Sigma) are exemplary nutrientsolutions. In addition, any of the media described in Ham and Wallace,(1979)Meth. Enzymol. 58:44; Barnes and Sato, (1980) Anal. Biochem.102:255; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 5,122,469 or4,560,655; International Publication Nos. WO 90/03430; and WO 87/00195;the disclosures of all of which are incorporated herein by reference,can be used as culture media. Any of these media can be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asgentamycin), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range) lipids (such aslinoleic or other fatty acids) and their suitable carriers, and glucoseor an equivalent energy source. In some embodiments the nutrient mediais serum-free media, a protein-free media, or a chemically definedmedia. Any other necessary supplements can also be included atappropriate concentrations that would be known to those skilled in theart.

In one embodiment, the mammalian host cell is a CHO cell and a suitablemedium contains a basal medium component such as a DMEM/HAM F-12 basedformulation (for composition of DMEM and HAM F12 media, see culturemedia formulations in American Type Culture Collection Catalogue of CellLines and Hybridomas, Sixth Edition, 1988, pages 346-349) with modifiedconcentrations of some components such as amino acids, salts, sugar, andvitamins, and optionally containing glycine, hypoxanthine, andthymidine; recombinant human insulin, hydrolyzed peptone, such asPrimatone HS or Primatone RL (Sheffield, England), or the equivalent; acell protective agent, such as Pluronic F68 or the equivalent pluronicpolyol; gentamycin; and trace elements.

The present invention provides a variety of media formulations that,when used in accordance with other culturing steps described herein,minimize, prevent or reverse metabolic imbalances in the culture thatwould lead to increased lactate and ammonium production.

A media formulation of the present invention that have been shown tohave beneficial effects on metabolic balance, cell growth and/orviability or on expression of polypeptide or protein comprise dextransulfate. One of ordinary skill in the art will understand that the mediaformulations of the present invention encompass both defined andnon-defined media.

Cell Culture Processes

Various methods of preparing mammalian cells for -production of proteinsor polypeptides by batch and fed-batch culture are well known in theart. A nucleic acid sufficient to achieve expression (typically a vectorcontaining the gene encoding the polypeptide or protein of interest andany operably linked genetic control elements) can be introduced into thehost cell line by any number of well-known techniques. Typically, cellsare screened to determine which of the host cells have actually taken upthe vector and express the polypeptide or protein of interest.Traditional methods of detecting a particular polypeptide or protein ofinterest expressed by mammalian cells include but are not limited toimmunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, SDS-PAGE, Western blots, enzyme-linkedimmunosorbent assay (ELISA), high performance liquid chromatography(HPLC) techniques, biological-activity assays and affinitychromatography. One of ordinary skill in the art will be aware of otherappropriate techniques for detecting expressed polypeptides or proteins.If multiple host cells express the polypeptide or protein of interest,some or all of the listed techniques can be used to determine which ofthe cells expresses that polypeptide or protein at the highest levels.

Once a cell that expresses the polypeptide or protein of interest hasbeen identified, the cell is propagated in culture by any of the varietyof methods well-known to one of ordinary skill in the art. The cellexpressing the polypeptide of interest is typically propagated bygrowing it at a temperature and in a medium, that is conducive to thesurvival, growth and viability of the cell. The initial culture volumecan be of any size, but is often smaller than the culture volume of theproduction bioreactor used in the final production of the polypeptide orprotein of interest, and frequently cells are passaged several times inbioreactors of increasing volume prior to seeding the productionbioreactor. The cell culture can be agitated or shaken to increaseoxygenation of the medium and dispersion of nutrients to the cells.Alternatively, or additionally, special sparging devices that are wellknown in the art can be used to increase and control oxygenation of theculture. In accordance with the present invention, one of ordinary skillin the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor, including butnot limited to pH, temperature, oxygenation, etc.

The cell density useful in the methods of the present invention can bechosen by one of ordinary skill in the art. In accordance with thepresent invention, the cell density can be as low as a single cell perculture volume. In some embodiments of the present invention, startingcell densities can range from about 2×10² viable cells per mL to about2×10³, 2×10⁴, 2×10⁵, 2×10⁶, 5×10⁶ or 10×10⁶ viable cells per mL andhigher.

In accordance with the present invention, a-cell culture size can be anyvolume that is appropriate for production of polypeptides. In oneembodiment, the volume of the cell culture is at least 500 liters. Inother embodiments, the volume or the production cell culture is 10, 50,100, 250, 1000, 2000, 2500, 5000, 8000, 10,000, 12,000 liters or more,or any volume in between. For example, a cell culture will be 10 to5,000 liters, 10 to 10,000 liters, 10 to 15,000 liters, 50 to 5,000liters, 50 to 10,000 liters, or 50 to 15,000 liters, 100 to 5,000liters, 100 to 10,000 liters, 100 to 15,000 liters, 500 to 5,000 liters,500 to 10,000 liters, 500 to 15,000 liters, 1,000 to 5,000 liters, 1,000to 10,000 liters, or 1,000 to 15,000 liters. Alternatively, a cellculture will be between about 500 liters and about 30,000 liters, about500 liters and about 20,000 liters, about 500 liters and about 10,000liters, about 500 liters and about 5,000 liters, about 1,000 titers andabout 30,000 liters, about 2,000 liters and about 30,000 liters, about3,000 liters and about 30,000 liters, about 5,000 liters and about30,000 liters, or about 10,000 liters and about 30,000 liters, or a cellculture will be at least about 500 liters, at least about 1,000 liters,at least about 2,000 liters, at least about 3,000 liters, at least about5,000 liters, at least about 10,000 liters, at least about 15,000liters, or at least about 20,000 liters.

One of ordinary skill in the art will be aware of and will be able tochoose a suitable culture size for use in practicing the presentinvention. The production bioreactor for the culture can be constructedof any material that is conducive to cell growth and viability that doesnot interfere with expression or stability of the produced polypeptideor protein.

The temperature of the cell culture will be selected based primarily onthe range of temperatures at which the cell culture remains viable. Forexample, during the initial growth phase, CHO cells grow well at 37° C.In general, most mammalian cells grow well within a range of about 25°C. to 42° C.

In one embodiment of the present invention, the temperature of theinitial growth phase is maintained at a single, constant temperature. Inanother embodiment, the temperature of the initial growth phase ismaintained within a range of temperatures. For example, the temperaturecan be steadily increased or decreased during the initial growth phase.Alternatively, the temperature can be increased or decreased by discreteamounts at various times during the initial, growth phase. One ofordinary skill in the art will be able to determine whether a single ormultiple temperature should be used, and whether the temperature shouldbe adjusted steadily or by discrete amounts.

The cells can be grown during the initial growth phase for a greater orlesser amount of time, depending on the needs of the practitioner andthe requirement of the cells themselves. In one embodiment, the cellsare grown for a period of time sufficient to achieve a viable celldensity that is a given percentage of the maximal viable cell densitythat the cells would eventually reach if allowed to grow undisturbed.For example, the cells can be grown for a period of time sufficient toachieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of maximalviable cell density.

In another embodiment the cells are allowed to grow for a defined periodof time. For example, depending on the starting concentration of thecell culture, the temperature at which the cells are grown, and theintrinsic growth rate of the cells, the cells can be grown for 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moredays. In some cases, the cells can be allowed to grow for a month ormore. In one embodiment, the growth phase is between about 1 day andabout 20 days, about 1 day and about 15 days, about 1 day and about 14days, about 1 day and about 13 days, about 1 day and about 12 days,about 1 day and about 11 days, about 1 day and about 10 days, about 1day and about 9 days, about 1 day and about 8 days, about 1 day andabout 7 days, about 1 day and about 6 days, about 1 day and about 5days, about 1 day and about 4 days, about 1 day and about 3 days, about2 clays and about 15 days, about 3 days and about 15 days, about 4 daysand about 15 days, about 5 days and about 15 days, about 6 days andabout 15 days, about 7 days and about 15 days, about 8 days and about 15days, about 9 days and about 15 days, about 10 days and about 15 days,about 2 days and about 20 days, about 3 days and about 20 days, about 4days and about 20 days, about 5 days and about 20 days, about 6 days andabout 20 days, about 7 days and about 20 days, about 8 days and about 20days, about 9 days and about 20 days, about 10 days and about 20 days,or about 10 days and about 20 days. In another embodiment, the growthphase is at least about 1 day, at least about 2 days, at least about 3days, at least about 4 days, at least about 5 days, at least about 6days, at least about 7 days, at least about 8 days, at least about 9clays, at least about 10 days, at least about 11 days, at least about 12days, at least about 15 days, or at least about 20 days. In a furtherembodiment, the growth phase is about 1 day, about 2 days, about 3 days,about 4 days, about 5 days, about 6 days, about 7 days, about 8 days,about 9 days, about 10 days, about 11 days, about 12 days, about 15days, or about 20 days.

The cells would be grown for 0 days in the production bioreactor iftheir growth in a seed bioreactor, at the initial growth phasetemperature, was sufficient that the viable cell density in theproduction bioreactor at the time of its inoculation is already at thedesired percentage of the maximal viable cell density. The practitionerof the present invention will be able to choose the duration of theinitial growth phase depending on polypeptide or protein productionrequirements and the needs of the cells themselves.

The cell culture can be agitated or shaken during the initial culturephase in order to increase oxygenation and dispersion of nutrients tothe cells. In accordance with the present invention, one of ordinaryskill in the art will understand that it can be beneficial to control orregulate certain internal conditions of the bioreactor during theinitial growth phase, including but not limited to pH, temperature,oxygenation, etc. For example, pH can be controlled by supplying anappropriate amount of acid or base and oxygenation can be controlledwith sparging devices that are well known in the art.

In one embodiment, at the end of the initial growth phase, at least oneof the culture conditions is shifted so that a second set of cultureconditions is applied. The shift in culture conditions can beaccomplished by a change in the temperature, pH, osmolality or chemicalinductant level of the cell culture. In one embodiment, the cultureconditions are shifted by shifting the temperature of the culture.

When shifting the temperature of the culture, the temperature shift canbe relatively gradual. For example, it can take several hours or days tocomplete the temperature change. Alternatively, the temperature shiftcan be relatively abrupt. For example, the temperature change can becomplete in less than several hours. Given the appropriate productionand control equipment, such as is standard in the commercial large-scaleproduction of polypeptides or proteins, the temperature change can evenbe complete within less than an hour.

The temperature of the cell culture in the subsequent growth phase willbe selected based primarily on the range of temperatures at which thecell culture remains viable and expresses recombinant polypeptides orproteins at commercially adequate levels. In general, most mammaliancells remain viable and express recombinant polypeptides or proteins atcommercially adequate levels within a range of about 25° C. to 42° C. Inone embodiment, mammalian cells remain viable and express recombinantpolypeptides or proteins at commercially adequate levels within a rangeof about 25° C. to 35° C. Those of ordinary skill in the art will beable to select appropriate temperature or temperatures in which to growcells, depending on the needs of the cells and the productionrequirements of the practitioner.

In accordance with the present invention, once the conditions of thecell culture have been shifted as discussed above, the cell culture ismaintained for a subsequent production phase under a second set ofculture conditions conducive to the survival and viability of the cellculture and appropriate for expression of the desired polypeptide orprotein at commercially adequate levels.

As discussed above, the culture can be shifted by shifting one or moreof a number of culture conditions including, but not limited to,temperature, pH, osmolality, and sodium butyrate levels. In oneembodiment, the temperature of the culture is shifted. According to thisembodiment, during the subsequent production phase, the culture ismaintained at a temperature or temperature range that is lower than thetemperature or temperature range of the initial growth phase. Forexample, during the subsequent production phase, CHO cells expressrecombinant polypeptides and proteins well within a range of 25° C. to35° C.

In accordance with the present invention, the cells can be maintained inthe subsequent production phase until a desired cell density orproduction titer is reached. In one embodiment, the cells are maintainedin the subsequent production phase until the titer to the recombinantpolypeptide or protein reaches a maximum. In other embodiments, theculture can be harvested prior to this point, depending on theproduction requirement of the practitioner or the needs of the cellsthemselves. For example, the cells can be maintained for a period oftime sufficient to achieve a viable cell density of 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percentof maximal viable cell density. In some cases, it is desirable to allowthe viable cell density to reach a maximum, and then allow the viablecell density to decline to some level before harvesting the culture. Inan extreme example, it can be desirable to allow the viable cell densityto approach or reach zero before harvesting the culture.

In another embodiment of the present invention, the cells are allowed togrow for a defined period of time during the subsequent productionphase. For example; depending on the concentration of the cell cultureat the start of the subsequent growth phase, the temperature at whichthe cells are grown, and the intrinsic growth rate of the cells, thecells can be grown for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or more days. In some cases, the cells can beallowed to grow for a month or more. The practitioner of the presentinvention will be able to choose the duration of the subsequentproduction phase depending on polypeptide or protein productionrequirements and the needs of the cells themselves.

In certain cases, it can be beneficial or necessary to supplement thecell culture during the growth and/or subsequent production phase withnutrients or other medium components that have been depleted ormetabolized by the cells. For example, it might be advantageous tosupplement the cell culture with nutrients or other medium componentsobserved to have been depleted. Alternatively, or additionally, it canbe beneficial or necessary to supplement the cell culture prior to thesubsequent production phase. As non-limiting examples, it can bebeneficial or necessary to supplement the cell culture with hormonesand/or other growth factors, particular ions (such as sodium, chloride,calcium, magnesium, and phosphate), buffers, vitamins, nucleosides ornucleotides, trace elements (inorganic compounds usually present at verylow final concentrations), amino acids, lipids, or glucose or otherenergy source.

These supplementary components, including the amino acids, can all beadded to the cell culture at one time, or they can be provided to thecell culture in a series of additions. In one embodiment of the presentinvention, the supplementary components are provided to the cell cultureat multiple times in proportional amounts. In another embodiment, it canbe desirable to provide only certain of the supplementary componentsinitially, and provide the remaining components at a later time. In yetanother embodiment of the present invention, the cell culture is fedcontinually with these supplementary components.

In accordance with the present invention, the total volume added to thecell culture should optimally be kept to a minimal amount. For example,the total volume of the medium or solution containing the supplementarycomponents added to the cell culture can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45 or 50% of the volume of the cell cultureprior to providing the supplementary components.

The cell culture can be agitated or shaken during the subsequentproduction phase in order to increase oxygenation and dispersion ofnutrients to the cells. In accordance with the present invention, one ofordinary skill in the art will understand that it can be beneficial tocontrol or regulate certain internal conditions of the bioreactor duringthe subsequent growth phase, including but not limited to pH,temperature, oxygenation, etc. For example, pH can be controlled bysupplying an appropriate amount of acid or base and oxygenation can becontrolled with sparging devices that are well known in the art.

In certain embodiments of the present invention, the practitioner canfind it beneficial or necessary to periodically monitor particularconditions of the growing cell culture. Monitoring cell cultureconditions allows the practitioner to determine whether the cell cultureis producing recombinant polypeptide or protein at suboptimal levels orwhether the culture is about to enter into a suboptimal productionphase.

In order to monitor certain cell culture conditions, it will benecessary to remove small aliquots of the culture for analysis. One ofordinary skill in the art will understand that such removal canpotentially introduce contamination into the cell culture, and will takeappropriate care to minimize the risk of such contamination.

As non-limiting example, it can be beneficial or necessary to monitortemperature, pH, cell density, cell viability, integrated viable celldensity, lactate levels, ammonium levels, osmolarity, or titer of theexpressed polypeptide or protein. Numerous techniques are well known inthe art that will allow one of ordinary skill in the art to measurethese conditions. For example, cell density can be measured using ahemocytometer, a Coulter counter, or Cell density examination (CEDEX).Viable cell density can be determined by staining a culture sample withTrypan blue. Since only dead cells take up the Trypan blue, viable celldensity can be determined by counting the total number of cells,dividing the number of cells that take up the dye by the total number ofcells, and taking the reciprocal. HPLC can be used to determine thelevels of lactate, ammonium or the expressed polypeptide or protein.Alternatively, the level of the expressed polypeptide or protein can bedetermined by standard molecular biology techniques such as Coomassiestaining of SDS-PAGE gels, Western blotting, Bradford assays, Lowryassays, Biuret assays, and UV absorbance. It can also be beneficial ornecessary to monitor the post-translational modifications of theexpressed polypeptide or protein, including phosphorylation andglycosylation.

The practitioner can also monitor the metabolic status of the cellculture, for example, by monitoring the glucose, lactate, ammonium, andamino acid concentrations in the cell culture, as well as by monitoringthe oxygen production or carbon dioxide production of the cell culture.For example, cell culture conditions can be analyzed by using NOVABioprofile 100 or 400 (NOVA Biomedical, Wash.). Additionally, thepractitioner can monitor the metabolic state of the cell culture bymonitoring the activity of mitochondria. In embodiment, mitochondrialactivity can be monitored by monitoring the mitochondrial membranepotential using Rhodamine 123. Johnson et al., 1980. P.N.A.S.77(2):990-994.

Isolation of Expressed Polypeptide

In general, it will typically be desirable to isolate and/or purifyproteins or polypeptides expressed according to the present invention.In one embodiment, the expressed polypeptide or protein is secreted intothe medium and thus cells and other solids can be removed, as bycentrifugation or filtering for example, as a first step in thepurification process.

Alternatively, the expressed polypeptide can be bound to the surface ofthe host cell. In this embodiment, the media is removed and the hostcells expressing the polypeptide or protein are lysed as a first step inthe purification process. Lysis of mammalian host cells can be achievedby any number of means well known to those of ordinary skill in the art,including physical disruption by glass beads and exposure to high pHconditions.

The polypeptide can be isolated and purified by standard methodsincluding, but not limited to, chromatography (e.g., ion exchange,affinity, size exclusion, and hydroxyapatite chromatography), gelfiltration, centrifugation, or differential solubility, ethanolprecipitation or by any other available technique for the purificationof proteins (See, e.g., Scopes, (eds.), Protein Expression: A PracticalApproach, Oxford Univ. Press, 1999; and Deutscher, M, P., Simon, M. I.,Abelson, J. N. (eds.). Guide to Protein Purification: Meth. Enzymol.182), Academic Press, 1997, all incorporated herein by reference. Forimmunoaffinity chromatography in particular, the protein can be isolatedby binding it to an affinity column comprising antibodies that wereraised against that protein and were affixed to a stationary support.Alternatively, affinity tags such as an influenza coat sequence,poly-histidine, or glutathione-S-transferase can be attached to theprotein by standard recombinant techniques to allow for easypurification by passage over the appropriate affinity column. Proteaseinhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin,pepstatin or aprotinin can be added at any or all stages in order toreduce or eliminate degradation of the polypeptide or protein during thepurification process. Protease inhibitors are particularly desired whencells must be lysed in order to isolate and purify the expressedpolypeptide or protein. One of ordinary skill in the art will appreciatethat the exact purification technique will vary depending on thecharacter of the polypeptide or protein to be purified, the character ofthe cells from which the polypeptide or protein is expressed, and thecomposition of the medium in which the cells were grown.

Pharmaceutical Compositions

A polypeptide of therapeutic interest, such as an antibody, or fragmentthereof, or virus can be formulated as a pharmaceutical composition foradministration to a subject, e.g., to treat or prevent a disorder ordisease.

Typically, a pharmaceutical composition includes a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents; and the like that arc physiologically compatible. Thecomposition can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (See e.g., Berge, S. M., etal. (1977) J. Pharm. Sci. 66:1-19). In one embodiment, a pharmaceuticalcomposition is an immunogenic composition comprising a virus produced inaccordance with methods described herein.

Pharmaceutical formulation is a well-established art, and is furtherdescribed, e.g., in Gennaro (ed.), Remington. The Science and Practiceof Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN:0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug DeliverySystems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN:0683305727); and Kibbe (ed,), Handbook of Pharmaceutical. ExcipientsAmerican Pharmaceutical Association, 3rd Ed. (2000) (ISBN: 091733096X).

The, pharmaceutical compositions can be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The form can depend on the intended mode of administration andtherapeutic application. Typically, compositions for the agentsdescribed herein are in the form of injectable or infusible solutions.

In one embodiment, an antibody is formulated with excipient materials,such as sodium chloride, sodium dibasic phosphate heptahydrate, sodiummonobasic phosphate, and a stabilizer. It can be provided, for example,in a buffered solution at a suitable concentration and can be stored at2-8° C.

Such compositions can be administered by a parenteral mode (e.g.,intravenous, subcutaneous, intraperitoneal, or intramuscular injection).The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for stablestorage at high concentration. Sterile injectable solutions can beprepared by incorporating an agent described herein in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating anagent described herein into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the methods of preparation are vacuumdrying and freeze drying that yield a powder of an agent describedherein plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the polypeptide can be prepared with a carrierthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known, See, e.g., Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York(1978).

The foregoing description is to be understood as being representativeonly and is not intended to be limiting. Alternative methods andmaterials for implementing the invention and also additionalapplications will be apparent to one of skill in the art, and areintended to be included within the accompanying claims.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory. Press (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription and Translation: B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y,; Gene Transfer Vectors forMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology Volumes-I-IV, D. M, Weir and C. C, Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General, principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ, Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, Mass.(1984); and Steward, M, W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al., (eds), Basic and Clinical—Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevier Amsterdam (1984), Kuby Immunology4th ed, Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne,H. Freemand & Co, (2000); Roitt, I., Brostoff, J. and Male D.,Immunology 6th ed, London: Mosby (2001); Abbas A. Abul, A. and Lichtman,A., Cellular and Molecular Immunology Ed 5, Elsevier Health SciencesDivision (2005); Kontermann and Dubel, Antibody Engineering, SpringerVerlan (2001); Sambrook and Russell, Molecular Cloning: A LaboratoryMammal, Cold Spring Harbor Press (2001); Lewin, Genes VIII, PrenticeHall (2003); Harlow and Lane, Antibodies; A Laboratory Manual, ColdSpring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer ColdSpring Harbor Press (2003).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

Reference will now be made to specific examples illustrating thedisclosure. It is to be understood that the examples are provided toillustrate exemplary embodiments and that no limitation to the scope ofthe disclosure is intended thereby.

EXAMPLES Example 1 Addition of Dextran Sulfate and Ferric CitrateMaintains Lactate Levels and Decreases Ammonium Production

Materials and Methods

Cell line: The cell line used in this study produced a neublastinpolypeptide. The cell line was constructed using DG44 adapted to grow inserum-free medium (Prentice, 2007).

Culture Medium: Basal and feed medium used for this experiment are bothproprietary in-house media that were previously described in Huang, 2010and Kshirsagar, 2012. Both media are chemically defined. Briefly thebasal medium CM3 was used for all, maintenance stages. A modifiedversion of CND, called CM3V2, with additional ferric citrate and dextransulfate, was used for the production stage. This medium containsglucose, amino acids, vitamins, minerals, and trace elements necessaryfor the robust cultivation of mammalian cells. Feed medium, is a moreconcentrated version of the basal medium with the nutritional contentoptimized to maximize growth and productivity. No lactate is present inthe feed medium. Again citrate is included in the feed medium as achelating agent but is present in feed medium at 2,4 InM citrate. Boththe basal medium and the feed medium comprised ferric citrate, Dextransulfate was included in feed medium between 0-10 g/L dextran sulfate.

Cell culture methods: Cells were thawed and maintained as in a previousreport (Kshirsagar, et al. 2012 Biotechnol. Bioeng. Huang, et al.,Biotechnol. Prog 26(5):1400-1410 (2010)). Basal medium for thaw andpassing was the same as in previous reports (Kshirsagar/Huang). Briefly,cells were thawed and maintained in 3 L shake flasks (Corning LifeSciences, Corning, N.Y.) with 1 L working volumes and were passagedevery 2 days. For maintenance cultures the incubator was Set at 36° C.and 5% CO₂.

Bioreactor culture conditions: Fed batch cultures were performed in S Lglass Applikon vessels using Finesse TruBio DV controllers (FinesseSolutions, San Jose, Calif.) with an initial working volume between2-2.5 L. Bioreaators were seeded at constant seed density of 4×105cells/ml. Concentrated feed medium was delivered on days 3, 5, and everyday following through harvest. Temperature was maintained at 36° C. andpH was controlled at 7.1+/−0.2 by the addition of either 1 M sodiumcarbonate or carbon dioxide. Dissolved oxygen was maintained at 30% byair and oxygen sparge using a drilled hole sparger. Agitation wasmaintained between 200-400 RPM throughout the culture to limit total gasflow, while an overlay was maintained between 0.005 and 0.04 vvm.

Offline analysis: Samples were taken on mast days and analyzed with avariety of equipment. Cell density and viability were measured using thestandard technique of trypan blue exclusion using a Cedex (RocheInnovatis AG, Germany), Glucose, lactate, ammonium, potassium and sodiumdata were collected using a NOVA Bioprofile 100 or 400 (NOVA Biomedical,Wash.).

In order to investigate the effect of dextran sulfate and ferric citrateon the lactate and the ammonium levels in cell culture, 0.25 g/L dextransulfate and 2.3 mM ferric citrate were added to the production medium onclay 0. In some cases, no additional dextran sulfate is provided. Insome cases, additional dextran sulfate is added via the feed.

In 902 using CM3 basal medium, lactate levels started at about 0.5 g/Lon day 0, peaked at about 2-2.5 g/L on day 3, and then rapidly decreasedto about 0.5 g/L from day 10 to day 14 (FIG. 1A), Lactate levels thenslightly increased again and remained at about 0.5-1/L between day 15and day 17, In the presence of dextran sulfate and ferric citrate. usingCM3v2 basal medium, lactate level in the 902 medium was well maintainedat about 2-2.5 g/L between day 3 and day 9, then decreased eventually toabout 1 g/L on day 17 (FIG. 1A). Similarly, lactate level in N65 cultureusing CM3 basal medium peaked at about 2.5 g/L on day 5, then rapidlydecreased to about 1 gE, on day 14, However, the presence of dextransulfate and ferric citrate almost sustained the lactate level in the N65medium at about 2.5-3 g/L between day 5 and day 17, with only a veryslight decrease from day 7 to day 16 (FIG. 1A).

Ammonium leveEq in the cell culture started at about 0.5 mivi on day 0in medium with or without dextran sulfate or ferric citrate (FIG. 1B),In 902, using CM3 basal medium, the ammonium level slightly increased toabout 3 mM on day 9, then dramatically climbed up to about 8 mM on day13, and reached about 9 mM on day 17. In the presence of dextran sulfateand ferric citrate, the ammonium production in the 902 medium wassignificantly reduced from day 0 to day 17, with between about 1 g/L andabout 3 g/L ammonium reduction from day 9 to day 14 (FIG. 1B). In N65using 0/13 basal medium, the ammonium level increased from 0.5 mM tonearly 4 mM on day 14, then slightly decreased to about 2 mM on day 17.In the presence of dextran sulfate and ferric citrate, the ammoniumproduction in the N65 medium was well maintained at or below 2 niM fromday 0 to day 17 (FIG. 1B).

Therefore, the addition of dextran sulfate and ferric citrate is able tomaintain lactate levels and decrease ammonium production in cellculture.

Example 2 Addition of Dextran Sulfate Stabilizes Viability of ShakeFlask Maintenance Culture

To investigate the effect of dextran sulfate on the viability of shakeflask maintenance culture, 0.1 g/L. dextran sulfate was added to acommercially available medium lacking dextran sulfate and CM3 HEKv1, arebalanced version of CM3 optimized for FIEK293 culture. While theviable cell density in medium comprising 0.1 g/L dextran sulfate wascomparable to that in medium with no dextran sulfate (FIG. 2A); thepresence of dextran sulfate greatly increased the percentage of viablecells (FIG. 2B). Cell viability in maintenance culture without dextransulfate went through frequent sudden decreases from day 0 to clay 32 andvaried dramatically between about 80% to about 95%, but the presence of0.1 g/L dextran sulfate effectively maintained, the percentage of cellviability above 95% at all the time points (FIG. 2B). Therefore,addition of dextran sulfate is able to stabilize viability of shakeflask maintenance culture.

Cell line: The cell line used in this study produced a Factor VIIIpolypeptide. The cell line was constructed using HEK 293 adapted to growin serum-free medium.

Culture medium: Basal and feed medium used for this experiment are bothmodified versions of proprietary in-house media that were previouslydescribed in Huang (2010) and Kshirsagar (2012) with rebalanced aminoacid and salt concentrations and named CM3.1-.ILK.y1. Both media arechemically defined. Briefly. the basal medium, CM3 I-IEKv I. was usedfor all maintenance stages unless otherwise noted. A modified version ofCM3 HEKv1, supplemented with dextran sulfate, was used for directcompulsion in both Maintenance and production culture. This mediumcontains glucose, amino acids, vitamins, minerals, and trace elementsnecessary for the robust cultivation of mammalian cells, Feed medium isa more concentrated version of the basal medium with the nutritionalcontent optimized to maximize growth and productivity. No lactate ispresent in the feed medium. Dextran sulfate was not included in feedmedium.

Cell culture methods: Cells were thawed and maintained as in a previousreport (Kshirsagar, et al, 2012 Biotechnol. Bioeng. Huang, et al,Biotechnology. Progress 26(5): 1400-1410 (2010)), Basal medium for thawand passing, CM3 HEKv1, was a modified version as that used in previousreports with rebalanced amino acid and salt concentrations(Kshirsagar/Huang) Briefly, cells were thawed and maintained in 1 Lshake flasks (Corning Life Sciences, Corning, N.Y.) with 0.2 L workingvolumes and were passaged every 2-3 days. For maintenance cultures theincubator was controlled at 37° C. and 10% CO₂.

Offline analysis: Samples were taken on most days and analyzed with avariety of equipment. Cell density and viability were measured using thestandard technique of trypan blue exclusion using a Cedex (RocheImiovatis AG, Germany). Glucose, lactate, ammonium, potassium, andsodium data were collected using a NOVA Bioprofile 100 or 400 (NOVABiomedical, Wash.).

Example 3 Addition of Dextran Sulfate Stabilizes Viability of BioreactorInoculum Train Culture

To investigate the effect of dextran sulfate on the viability ofbioreactor inoculum train culture, 0.1 g/L dextran sulfate was added toCM3 HEKv1 on day 0. While the presence of dextran sulfate did not affectthe viable cell density (FIG. 3A), it effectively maintained thepercentage of viable cells in the bioreactor inoculum train culture(FIG. 3B), Cell viability in inoculum train culture without dextransulfate dropped from about 85% on day 0 to below 60% on day 7, and wasbetween about 60% and about 75% after day 7, but addition of 0.1 g/Ldextran sulfate effectively maintained the percentage of cell viabilityat about 95% at all the time points 3B). Therefore, addition of dextransulfate is able to stabilize viability of bioreactor inoculum trainculture.

Bioreactor culture conditions: Fed batch cultures were performed M 5 Lglass Applikon vessels using Finesse TruBio DV controllers (FinesseSolutions, San Jose, Calif.) with an initial working volume between2-2.5 L. Bioreactors were seeded at constant seed density of 4×10cells/mi. Temperature was maintained at 37° C. and pH was controlled at7.0.11-0.3 by the addition of either 1 M sodium carbonate or carbondioxide, Dissolved oxygen was maintained at 50% by air and oxygen spargeusing a drilled hole sparger. Agitation was maintained at 125 RPMthroughout the culture to limit total gas flow, while an overlay wasmaintained between 0.005 and 0.04 vvm.

Example 4 Dextran Sulfate Containing Inoculum was Enough to StabilizeEarly Stage Culture Viability in Production Bioreactors

To further investigate whether the amount of dextran sulfate containedin the inoculum culture was able to stabilize cell viability inproduction bioreactors, bioreactor culture was inoculated with inoculumculture containing 0.1 g/L dextran sulfate on day 0, and no additionaldextran sulfate was added in subsequent feed medium, The presence ofdextran sulfate in the production bioreactor culture was able tostabilize both viable cell density and cell viability for two more days(FIGS. 4A-4B). In bioreactor culture without dextran sulfate, bothviable cell density and cell viability sharply decreased after day 12,but the presence of dextran sulfate postponed this decrease to day 14(FIG. 4B). In addition, the presence of dextran sulfate also greatlymaintained the cell viability above 95% from day 0 to day 6, when theviability in dextran sulfate free culture varied between 80% to about90% (FIG. 4B). Therefore, inoculation of production culture usingdextran sulfate containing inoculum is enough to stabilize early stageculture viability.

Bioreactor culture conditions: Fed batch cultures were performed in 5 Lglass Applikon vessels using Finesse TruBio DV controllers (FinesseSolutions, San Jose, Calif.) with an initial working volume between2-2.5 L. Bioreactors were seeded at constant seed density. of 5×10⁵cells/ml. Concentrated feed medium was delivered on day 3, and every dayfollowing through harvest. Temperature was maintained at 35.5° C. and pHwas controlled at 7.2+1-0.1 by the addition of either 1 M sodiumcarbonate or carbon dioxide. Dissolved oxygen was maintained at 30% byair and oxygen sparge using a drilled hole sparger. Agitation wasmaintained between 200-400 RPM throughout the culture to limit total gasflow, while an overlay was maintained between 0.005 and 0.04 vvm.

Offline analysis: Samples were taken on most days and analyzed with avariety of equipment. Cell density and viability were measured using thestandard technique of trypan blue exclusion using a Cedex (RocheInnovatis AG, Germany). Glucose, lactate, ammonium, potassium and sodiumdata were collected using a NOVA Bioprofile 100 or 400 (NOVA Biomedical,Wash.).

Example 5 Production of Neublastin Antibody in CHO Cell Cultures

The DNA encoding the heavy and light chains of the murine IgG1anti-neublastin monoclonal antibody (FIG. 8) was cloned into aeukaryotic expression vector plasmid bXLTBR.9 containing thecytomegalovirus (CMV) intermediate early promoter (pGV90). Cloning wasfollowed by transfection of the P3B3-containing plasmid intodihydrofolate reductase (DHFR)-deficient DG44i Chinese hamster ovary(CHO) host cells (Cell Line Ref: 13805-32 (Thermo Fisher, Waltham,Mass.) (Urlaub and Chasin (1980) P.N.A.S 77:4216-4221) using the FuGene6 transfection reagent (Roche Applied Sciences, Indianapolis Ind.).

The anti-neublastin mAb-producing clonal cell line (EAG2659/2660 Clone 8DG44i) was identified by iterative screening of transfected cells forhigh specific productivity by flow-assisted cell sorting (FACS) using ananti-human IgG1 monoclonal antibody (Brezinsky et al., (2003).

This cell line was grown in a neomycin selection medium containing 40μg/ml G418 (gentamicin, Thermo Fisher) or in a growth medium suitablefor CHO cell culture (e.g., in shaker flasks at 36.5° C. with 5% CO₂.When cell densities reached 3×10⁶ and 4×10⁶ cells/ml, cells weretransferred to a production vessel seeded with 2×10⁵ cells/ml and gownat 36.5° C. in 50% atmosphere in dissolved oxygen, with a pH set to 7.4.On day 3, with a density of 2×10⁵ cells/ml, the vessel was fed with 5%starting culture volume. On day 5, with a density of 4×10⁶-5×10⁶cells/ml, the temperature was dropped to 28° C. Cells were harvested bycentrifugation when the viable cell density had dropped to about 88%(day 18). The conditioned medium was centrifuges at over 1000 g for 20min and then filtered through a 0.2 μm filter to remove cells and toclarify. The clarified medium was then concentrated about 6× on aPrescale tangential flow 6 ft² 30 k mol. wt. cutoff cartridge(Millipore, Burington, Mass.). The concentrate supernatant can be storedfor over 1 year at −80° C.

The concentrated cell supernatant was brought to 3 M NaCl/1.5 M glycine,pH 8.9 (Single Stranded DNA (SS) Binding Buffer, Thermo FisherScientific) and loaded onto an recombinant Protein A Sepharose (recProASeph) 4FF column (G.E. Lifesciences, Chicago, Ill.). The column waswashed with about 7 column volumes (CV) of SS Binding Buffer at 5 CV of3 M NaCl/25 mM NaPO₄, pH 8.6. The protein was then eluded off the columnwith about 3.5 CV of 2 mm NaPO₄/100 mM NaCl, pH 2.8. The pooled eluatewas then dialyzed into 20 CV of PBS (20 mM NaPO₄/50 mM NaCl, pH 7.1)four times for a minimum of 8 hr each time. After dialysis, the materialwas 0.2 μm sterile-filtered and analyzed by size exclusionchromatography (SEC) on a Superdex200 (GE Healthcare Life Sciences,Chicago, Ill.) (1 cm×20 cm). FIG. 6 shows that the antibody (P3B3)eluted as an 158 k peak. The peak material was run on 4%-20% SDS-PAGE(FIG. 7) and tested for endotoxin. The amino acid sequence of the heavy(SEQ ID NO:2) and light (SEQ ID NO:4) chains of the antibody are shownin FIGS. 5B-5D.

EQUIVALENTS

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and, described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A cell culture comprising a mammalian cell line genetically modified to express, and which expresses, a neublastin antibody polypeptide, or a fragment thereof, in a cell culture medium, wherein the neublastin antibody polypeptide comprises: three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises amino acid residues 27 to 35 of SEQ ID NO:2, HCDR2 comprises amino acid residues 47 to 61 of SEQ ID NO:2, and HCDR3 comprises amino acid residues 97 to 111 of SEQ ID NO:2, and three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises amino acid residues 27 to 34 of SEQ ID NO:4, LCDR2 comprises amino acid residues 46 to 56 of SEQ ID NO:4, and LCDR3 comprises amino acid residues 89 to 96 of SEQ ID NO:4.
 2. The cell culture of claim 1, wherein the neublastin antibody polypeptide comprises SEQ ID NO: 2, or a fragment thereof.
 3. The cell culture of claim 1, wherein the neublastin antibody polypeptide comprises SEQ ID NO: 4, or a fragment thereof.
 4. The cell culture of claim 1, wherein the neublastin antibody polypeptide comprises SEQ ID NO: 4, or a fragment thereof, and wherein the neublastin antibody polypeptide comprises SEQ ID NO: 2, or a fragment thereof.
 5. The cell culture of claim 1, wherein the mammalian cell line is a Chinese Hamster Ovary (CHO) cell line.
 6. The mammalian cell culture of claim 1, wherein the cells have been adapted to grow in a serum-free medium, an animal protein-free medium, or a chemically defined medium.
 7. The cell culture of claim 1, wherein the mammalian cells have been genetically modified with a polynucleotide encoding the neublastin antibody polypeptide, or a fragment thereof.
 8. The mammalian cell culture of claim 1, wherein the culture is a perfusion culture or is a fed batch culture.
 9. The mammalian cell culture of claim 1, wherein the medium is a serum-free medium, AN animal protein-free medium, or a chemically defined medium.
 10. A neublastin antibody polypeptide produced in a large-scale mammalian cell culture, the culture comprising mammalian cells genetically modified to express, and which express, the neublastin antibody polypeptide, or a fragment thereto, in a mammalian cell culture, wherein the neublastin antibody polypeptide comprises: three heavy chain complementarity determining regions (HCDRs), wherein HCDR1 comprises amino acid residues 27 to 35 of SEQ ID NO:2, HCDR2 comprises amino acid residues 47 to 61 of SEQ ID NO:2, and HCDR3 comprises amino acid residues 97 to 111 of SEQ ID NO:2, and three light chain complementarity determining regions (LCDRs), wherein LCDR1 comprises amino acid residues 27 to 34 of SEQ ID NO:4, LCDR2 comprises amino acid residues 46 to 56 of SEQ ID NO:4, and LCDR3 comprises amino acid residues 89 to 96 of SEQ ID NO:4.
 11. The neublastin antibody polypeptide of claim 10, comprising a heavy chain polypeptide comprising SEQ ID NO: 2, or a fragment thereof.
 12. The neublastin antibody polypeptide of claim 10, comprising a light chain polypeptide comprising SEQ ID NO: 4, or a fragment thereof.
 13. The neublastin antibody polypeptide of claim 10, comprising a light chain polypeptide comprising SEQ ID NO: 4, or a fragment thereof, and a heavy chain polypeptide comprising SEQ ID NO: 2, or a fragment thereof.
 14. The neublastin antibody polypeptide of claim 10, wherein the mammalian cell culture comprises CHO cells expressing the neublastin antibody polypeptide, or a fragment thereof.
 15. The neublastin antibody polypeptide of claim 10, wherein the cells have been adapted to grow in serum-free medium, an animal protein-free medium, or a chemically defined medium.
 16. The neublastin antibody polypeptide, or fragment thereof, of claim 10, which has been isolated from the mammalian cell culture.
 17. A pharmaceutical formulation comprising the neublastin antibody of claim 16 in a pharmaceutically acceptable carrier. 